U.S. patent application number 12/704948 was filed with the patent office on 2010-06-10 for interferon alpha receptor i antibodies and their use.
Invention is credited to Josephine M. Cardarelli, Mohan Srinivasan, Alison Witte.
Application Number | 20100143369 12/704948 |
Document ID | / |
Family ID | 35782299 |
Filed Date | 2010-06-10 |
United States Patent
Application |
20100143369 |
Kind Code |
A1 |
Cardarelli; Josephine M. ;
et al. |
June 10, 2010 |
Interferon Alpha Receptor I Antibodies And Their Use
Abstract
The present invention provides isolated human monoclonal
antibodies that bind to IFNAR-1 and that are capable of inhibiting
the biological activity of Type I interferons. Immunoconjugates,
bispecific molecules and pharmaceutical compositions comprising the
antibodies of the invention are also provided. The invention also
provides methods for inhibiting Type I interferon-mediated
disorders using the antibodies of the invention, including methods
for treating autoimmune disorders, transplant rejection or Graft
Versus Host Disease using the antibodies of the invention.
Inventors: |
Cardarelli; Josephine M.;
(San Carlos, CA) ; Witte; Alison; (Scotts Valley,
CA) ; Srinivasan; Mohan; (San Jose, CA) |
Correspondence
Address: |
BAKER BOTTS L.L.P.
30 ROCKEFELLER PLAZA, 44th Floor
NEW YORK
NY
10112-4498
US
|
Family ID: |
35782299 |
Appl. No.: |
12/704948 |
Filed: |
February 12, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11157494 |
Jun 20, 2005 |
7662381 |
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12704948 |
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60581747 |
Jun 21, 2004 |
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Current U.S.
Class: |
424/142.1 |
Current CPC
Class: |
A61P 19/02 20180101;
A61P 29/00 20180101; A61P 3/10 20180101; A61P 1/00 20180101; C07K
2317/76 20130101; A61P 17/16 20180101; A61P 19/04 20180101; A61P
25/00 20180101; A61P 13/12 20180101; C07K 2317/56 20130101; A61P
37/06 20180101; C07K 16/2866 20130101; A61P 31/18 20180101; C07K
2317/31 20130101; A61P 35/00 20180101; A61P 43/00 20180101; A61P
17/02 20180101; A61P 25/28 20180101; A61P 17/06 20180101; C07K
2317/21 20130101; A61P 5/48 20180101; A61P 37/02 20180101; C07K
2317/92 20130101; C07K 2317/565 20130101; A61P 5/14 20180101; C07K
2317/74 20130101; A61P 1/04 20180101; A61P 37/00 20180101 |
Class at
Publication: |
424/142.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 37/06 20060101 A61P037/06 |
Claims
1. A method of treating a type-I interferon-mediated disease or
disorder in a subject in need of treatment comprising administering
to the subject an isolated human monoclonal antibody, or an
antigen-binding portion thereof, that specifically binds to human
interferon alpha receptor 1 (IFNAR-I), wherein the antibody
inhibits the biological activity of IFN-.beta. and does not bind
the same epitope as mouse monoclonal antibody 64G12 (ECACC Deposit
No. 92022605), such that the type-I interferon mediated disease in
the subject is treated.
2. A method of treating a type-I interferon-mediated disease or
disorder in a subject in need of treatment comprising administering
to the subject an effective amount of an isolated human monoclonal
antibody, or an antigen-binding portion thereof, that
cross-competes for binding to human interferon alpha receptor I
(IFNAR-I) with a reference antibody, wherein the reference antibody
is selected from the group consisting of: a) an antibody comprising
a heavy chain variable region comprising the amino acid sequence of
SEQ ID NO: 25; and a light chain variable region comprising the
amino acid sequence of SEQ ID NO: 29; b) an antibody comprising a
heavy chain variable region comprising the amino acid sequence of
SEQ ID NO: 26; and a light chain variable region comprising the
amino acid sequence of SEQ ID NO: 30; c) an antibody comprising a
heavy chain variable region comprising the amino acid sequence of
SEQ ID NO: 27; and a light chain variable region comprising the
amino acid sequence of SEQ ID NO: 31; and d) an antibody comprising
a heavy chain variable region comprising the amino acid sequence of
SEQ ID NO: 28; and a light chain variable region comprising the
amino acid sequence of SEQ ID NO: 32.
3. The method of claim 1 or 2, wherein the type-I
interferon-mediated disease is an interferon alpha-mediated
disease.
4. The method of claim 1 or 2, wherein the disease or disorder is
systemic lupus erythematosus.
5. The method of claim 1 or 2, wherein the disease or disorder is
selected from the group consisting of insulin dependent diabetes
mellitus, inflammatory bowel disease, multiple sclerosis,
psoriasis, autoimmune thyroiditis, rheumatoid arthritis and
glomerulonephritis.
6. The method of claim 1 or 2, wherein the disease or disorder is
HIV infection or AIDS.
7. The method of claim 1 or 2, wherein the disease or disorder is
transplant rejection or graft versus host disease.
8. The method of claim 1 or 2, wherein the anti-type 1 interferon
receptor antibody is administered by a route selected from the
group consisting of subcutaneously, intramuscularly, intradermally,
intraperitoneally, and intravenously.
9. The method of claim 1 or 2, wherein the anti-type 1 interferon
receptor antibody is administered intravenously.
10. The method of claim 1 or 2, wherein the anti-type 1 interferon
receptor antibody is administered in a dosage range of between
about 1 mg/kg body weight and 10 mg/kg body weight.
11. The method of claim 10, wherein the anti-type 1 interferon
receptor antibody is administered in a dosage selected from the
group consisting of: about 0.3 mg/kg body weight, about 1 mg/kg
body weight, about 3 mg/kg body weight, about 5 mg/kg body weight
and about 10 mg/kg body weight.
12. The method of claim 1 or 2, wherein the anti-type 1 interferon
receptor antibody is administered at a frequency selected from the
group consisting of once per week, once every two weeks, once every
three weeks, once every four weeks, once a month, once every 3
months and once every three to 6 months.
13. The method of claim 1 or 2, wherein the anti-type 1 interferon
receptor antibody is administered every four weeks for six dosages,
followed by once every three months.
14. The method of claim 1 or 2, wherein the anti-type 1 interferon
receptor antibody is administered once at about 3 mg/kg body weight
followed by about 1 mg/kg body weight every three weeks.
15. The method of claim 1, wherein the isolated human monoclonal
antibody, or antigen-binding portion thereof, comprises: (a) a
human heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO: 1; (b) a human heavy chain variable region
CDR2 comprising the amino acid sequence of SEQ ID NO:5; (c) a human
heavy chain variable region CDR3 comprising the amino acid sequence
of SEQ ID NO:9; (d) a human light chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 13; (e) a human
light chain variable region CDR2 comprising the amino acid sequence
of SEQ ID NO: 17; and (f) a human light chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO:21.
16. The method of claim 1, wherein the isolated human monoclonal
antibody, or antigen-binding portion thereof, comprises: (a) a
human heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO:2; (b) a human heavy chain variable region
CDR2 comprising the amino acid sequence of SEQ ID NO:6; (c) a human
heavy chain variable region CDR3 comprising the amino acid sequence
of SEQ ID NO: 10; (d) a human light chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 14; (e) a human
light chain variable region CDR2 comprising the amino acid sequence
of SEQ ID NO: 18; and (f) a human light chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO:22.
17. The method of claim 1, wherein the isolated human monoclonal
antibody, or antigen-binding portion thereof, comprises: (a) a
human heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO:3; (b) a human heavy chain variable region
CDR2 comprising the amino acid sequence of SEQ ID NO:7; (c) a human
heavy chain variable region CDR3 comprising the amino acid sequence
of SEQ ID NO: 11; (d) a human light chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 15; (e) a human
light chain variable region CDR2 comprising the amino acid sequence
of SEQ ID NO: 19; and (f) a human light chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO:23.
18. The method of claim 1, wherein the isolated human monoclonal
antibody, or antigen-binding portion thereof, comprises: (a) a
human heavy chain variable region CDR1 comprising the amino acid
sequence of SEQ ID NO:4; (b) a human heavy chain variable region
CDR2 comprising the amino acid sequence of SEQ ID NO:8; (c) a human
heavy chain variable region CDR3 comprising the amino acid sequence
of SEQ ID NO: 12; (d) a human light chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 16; (e) a human
light chain variable region CDR2 comprising the amino acid sequence
of SEQ ID NO:20; and (f) a human light chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO:24.
19. The method of claim 1, wherein the isolated human monoclonal
antibody, or antigen-binding portion thereof, comprises: (a) a
human heavy chain variable region comprising the amino acid
sequence of SEQ ID NO:25; and (b) a human light chain variable
region comprising the amino acid sequence of SEQ ID NO:29.
20. The method of claim 1, wherein the isolated human monoclonal
antibody, or antigen-binding portion thereof, comprises: (a) a
human heavy chain variable region comprising the amino acid
sequence of SEQ ID NO:26; and (b) a human light chain variable
region comprising the amino acid sequence of SEQ ID NO:30.
21. The method of claim 1, wherein the isolated human monoclonal
antibody, or antigen-binding portion thereof, comprises: (a) a
human heavy chain variable region comprising the amino acid
sequence of SEQ ID NO:27; and (b) a human light chain variable
region comprising the amino acid sequence of SEQ ID NO:31.
22. The method of claim 1, wherein the isolated human monoclonal
antibody, or antigen-binding portion thereof, comprises: (a) a
human heavy chain variable region comprising the amino acid
sequence of SEQ NO:28; and (b) a human light chain variable region
comprising the amino acid sequence of SEQ ID NO:32.
23. The method of claim 2, wherein the reference antibody is an
antibody comprising a heavy chain variable region comprising the
amino acid sequence of SEQ ID NO: 28; and a light chain variable
region comprising the amino acid sequence of SEQ ID NO: 32.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/157,494, filed on Jun. 20, 2005 and issued as U.S. Pat.
No. 7,662,381, which claims priority to U.S. provisional
application No. 60/581,747, filed on Jun. 21, 2004, the contents of
which are hereby incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] Type I interferons (IFN) (IFN-.alpha., IFN-.beta.,
IFN-.omega., IFN-.tau.) are a family of structurally related
cytokines having antiviral, antitumor and immunomodulatory effects
(Hardy et al. (2001) Blood 97:473; Cutrone and Langer (2001) J.
Biol. Chem. 276:17140). The human IFN.alpha. locus includes two
subfamilies. The first subfamily consists of 14 non allelic genes
and 4 pseudogenes having at least 80% homology. The second
subfamily, .alpha.II or omega (.omega.), contains 5 pseudogenes and
1 functional gene which exhibits 70% homology with the IFN.alpha.
genes (Weissmann and Weber (1986) Prog. Nucl. Acid Res. Mol. Biol.,
33:251-300). The subtypes of IFN.alpha. have different specific
activities but they possess the same biological spectrum (Streuli
et al. (1981) Proc. Natl. Acad. Sci. USA 78:2848) and have the same
cellular receptor (Agnet M. et al. in "Interferon 5" Ed. I. Gresser
p. 1-22, Academic Press, London 1983).
[0003] The interferon .beta. (IFN .beta.) is encoded by a single
gene which has approximately 50% homology with the IFN.alpha.
genes.
[0004] Gamma interferon, which is produced by activated
lymphocytes, does not possess any homology with the alpha/beta
interferons and it does not react with their receptor.
[0005] All human type I interferons bind to a cell surface receptor
(IFN alpha receptor, IFNAR) consisting of two transmembrane
proteins, IFNAR-1 and IFNAR-2 (Uze et. al. (1990) Cell 60:225;
Novick et al. (1994) Cell 77:391). IFNAR-1 is essential for high
affinity binding and differential specificity of the IFNAR complex
(Cutrone, 2001, supra). While functional differences for each of
the type I IFN subtypes have not been identified it is thought that
each may exhibit different interactions with the IFNAR receptor
components leading to potentially diverse signaling outcomes (Cook
et al. (1996) J. Biol. Chem. 271:13448). In particular, studies
utilizing mutant forms of IFNAR1 and IFNAR2 suggested that alpha
and beta interferons signal differently through the receptor by
interacting differentially with respective chains (Lewerenz et al.
(1998) J. Mol. Biol. 282:585).
[0006] Early functional studies of type I IFNs focused on innate
defense against viral infections (Haller et al. (1981) J. Exp. Med.
154:199; Lindenmann et al. (1981) Methods Enzymol. 78:181). More
recent studies, however, implicate type I IFNs as potent
immunoregulatory cytokines in the adaptive immune response.
Specifically, type I IFNs have been shown to facilitate
differentiation of naive T cells along the Thi pathway (Brinkmann
et al. (1993) J. Exp. Med. 178:1655), to enhance antibody
production (Finkelman et al. (1991) J. Exp. Med. 174:1179) and to
support the functional activity and survival of memory T cells
(Santini et al. (2000) J. Exp. Med. 191:1777; Tough et al. (1996)
Science 272:1947).
[0007] Recent work by a number of groups suggests that IFN-.alpha.
may enhance the maturation or activation of dendritic cells (DCs)
(Santini, et al. (2000) J. Exp. Med. 191:1777; Luft et al. (1998)
J. Immunol. 161:1947; Luft et al. (2002) Int. Immunol. 14:367;
Radvanyi et al. (1999) Scand. J. Immunol. 50:499). Furthermore,
increased expression of type I interferons has been described in
numerous autoimmune diseases (Foulis et al. (1987) Lancet 2:1423;
Hooks et al. (1982) Arthritis Rheum. 25:396; Hertzog et al. (1988)
Clin. Immunol. Immunopathol. 48:192; Hopkins and Meager (1988)
Clin. Exp. Immunol. 73:88; Arvin and Miller (1984) Arthritis Rheum.
27:582). The most studied examples of this are insulin-dependent
diabetes mellitus (IDDM) (Foulis (1987) supra) and systemic lupus
erythematosus (SLE) (Hooks (1982) supra), which are associated with
elevated levels of IFN-.alpha., and rheumatoid arthritis (RA)
(Hertzog (1988), Hopkins and Meager (1988), Arvin and Miller
(1984), supra) in which IFN-.beta. may play a more significant
role.
[0008] Moreover, administration of interferon a has been reported
to exacerbate underlying disease in patients with psoriasis and
multiple sclerosis and to induce an SLE like syndrome in patients
without a previous history of autoimmune disease. Interferon
.alpha. has also been shown to induce glomerulonephritis in normal
mice and to accelerate the onset of the spontaneous autoimmune
disease of NZB/W mice. Further, IFN-.alpha. therapy has been shown
in some cases to lead to undesired side effects, including fever
and neurological disorders. Hence there are pathological situations
in which inhibition of Type I IFN activity may be beneficial to the
patient and a need exists for agents effective in inhibiting Type I
IFN activity.
SUMMARY OF THE INVENTION
[0009] The present invention provides isolated human monoclonal
antibodies that bind to IFNAR-1 and inhibit the biological activity
of type I interferon, preferably multiple type I interferons.
Furthermore, the antibodies do not bind to the same epitope as the
murine anti-IFNAR-1 antibody, 64G12.
[0010] In one aspect, the, invention pertains to an isolated human
antibody, or antigen binding portion thereof, wherein the antibody
specifically binds to IFNAR-1 and exhibits one or more of the
following properties:
[0011] a) binds to IFNAR-1 with a K.sub.D of 1.times.10.sup.-7 M or
greater affinity;
[0012] b) inhibits the biological activity of multiple Type I
interferons;
[0013] c) inhibits the activity of IFN .alpha. 2b in a Daudi cell
proliferation assay;
[0014] d) inhibits the activity of IFN omega in a Daudi cell
proliferation assay;
[0015] e) inhibits IP-10 secretion by peripheral blood mononuclear
cells induced by IFN .alpha. 2b;
[0016] f) inhibits IP-10 secretion by peripheral blood mononuclear
cells induced by IFN omega;
[0017] g) inhibits dendritic cell development mediated by Systemic
Lupus Erythematosus plasma; and
[0018] h) binds to a different epitope than murine monoclonal
antibody 64G12 (ECACC Deposit No. 92022605).
[0019] Preferred antibodies of the invention specifically bind to
human interferon alpha receptor 1 and bind with a K.sub.D of
1.times.10.sup.-8 M or greater affinity, or 1.times.10.sup.-9 M or
greater affinity, or 5.times.10.sup.-10 M or greater affinity or
2.times.10.sup.-10 M or greater affinity
[0020] In one aspect, the invention pertains to an isolated
monoclonal antibody, or an antigen-binding portion thereof,
comprising a heavy chain variable region that is the product of or
derived from a human V.sub.H 4-34 or 5-51 gene, wherein the
antibody specifically binds to human interferon alpha receptor 1.
In another aspect, the invention pertains to an isolated monoclonal
antibody, or an antigen-binding portion thereof, comprising a light
chain variable region that is the product of or derived from a
human V.sub.K L18 or A27 gene, wherein the antibody specifically
binds to human interferon alpha receptor 1. In yet another aspect,
the invention pertains to an isolated human monoclonal antibody, or
antigen-binding portion thereof, comprising:
[0021] (a) a heavy chain variable region that is the product of or
derived from a human V.sub.H 4-34 or 5-51 gene; and
[0022] (b) a light chain variable region that is the product of or
derived from a human Vk L18 or A27 gene;
[0023] wherein the antibody specifically binds to human interferon
alpha receptor 1. In preferred embodiments, the antibody comprises
a heavy chain variable region of a human V.sub.H 4-34 gene and a
light chain variable region of a human V.sub.K L18 gene or the
antibody comprises a heavy chain variable region of a human V.sub.H
5-51 gene and a light chain variable region of a human V.sub.K A27
gene.
[0024] In another aspect, the invention provides an isolated human
monoclonal antibody, or antigen-binding portion thereof,
comprising:
[0025] a human heavy chain variable region comprising CDR1, CDR2,
and CDR3 sequences; and a human light chain variable region
comprising CDR1, CDR2, and CDR3 sequences, wherein:
[0026] (a) the human heavy chain variable region CDR3 sequence
comprises an amino acid sequence selected from the group consisting
of amino acid sequences of SEQ ID NO: 9, 10, 11, and 12, and
conservative modifications thereof;
[0027] (b) the human light chain variable region CDR3 sequence
comprises an amino acid sequence selected from the group consisting
of amino acid sequence of SEQ ID NO:21, 22, 23, and 24, and
conservative modifications thereof;
[0028] (c) the antibody specifically binds human interferon alpha
receptor 1 with a binding affinity of at least 1.times.10.sup.-8 M
or greater affinity; and
[0029] (d) the antibody inhibits the biological activity of at
least one Type I interferon.
[0030] Preferably, the human heavy chain variable region CDR2
sequence comprises an amino acid sequence selected from the group
consisting of amino acid sequences of SEQ ID NO: 5, 6, 7, and 8,
and conservative modifications thereof; and the human light chain
variable region CDR2 sequence comprises an amino acid sequence
selected from the group consisting of amino acid sequences of SEQ
ID NO: 17, 18, 19, and 20, and conservative modifications thereof
Preferably, the human heavy chain variable region CDR1 sequence
comprises an amino acid sequence selected from the group consisting
of amino acid sequences of SEQ ID NO: 1, 2, 3, and 4, and
conservative modifications thereof; and the human light chain
variable region CDR1 sequence comprises an amino acid sequence
selected from the group consisting of amino acid sequences of SEQ
ID NO: 13, 14, 15, and 16, and conservative modifications
thereof.
[0031] In another aspect, the invention pertains to an isolated
human monoclonal antibody, or antigen-binding portion thereof,
comprising a human heavy chain variable region and a human light
chain variable region, wherein:
[0032] (a) the human heavy chain variable region comprises an amino
acid sequence that is at least 80% homologous to an amino acid
sequence selected from the group consisting of SEQ ID NO: 25, 26,
27, and 28;
[0033] (b) the human light chain variable region comprises an amino
acid sequence that is at least 80% homologous to an amino acid
sequence selected from the group consisting of SEQ ID NO: 29, 30,
31, and 32;
[0034] (c) the antibody specifically binds human interferon alpha
receptor 1 with a binding affinity of at least 1.times.10.sup.-8 M
or greater affinity; and
[0035] (d) the antibody inhibits the biological activity of at
least one Type I interferon.
[0036] Preferred antibodies of the invention include isolated human
monoclonal antibodies, or antigen-binding portions thereof,
comprising:
[0037] (a) a human heavy chain variable region CDR1 comprising an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 1, 2, 3, and 4;
[0038] (b) a human heavy chain variable region CDR2 comprising an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 5, 6, 7, and 8;
[0039] (c) a human heavy chain variable region CDR3 comprising an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 9, 10, 11, and 12;
[0040] (d) a human light chain variable region CDR1 comprising an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 13, 14, 15, and 16;
[0041] (e) a human light chain variable region CDR2 comprising an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 17, 18, 19, and 20; and
[0042] (f) a human light chain variable region CDR3 comprising an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 21, 22, 23, and 24;
[0043] wherein the antibody specifically binds human interferon
alpha receptor 1 with a binding affinity of at least
1.times.10.sup.-8 M or greater affinity.
[0044] Preferred combinations of CDR regions include the
following:
[0045] (a) a human heavy chain variable region CDR1 comprising SEQ
ID NO:1;
[0046] (b) a human heavy chain variable region CDR2 comprising SEQ
ID NO:5;
[0047] (c) a human heavy chain variable region CDR3 comprising SEQ
ID NO:9;
[0048] (d) a human light chain variable region CDR1 comprising SEQ
ID NO:13;
[0049] (e) a human light chain variable region CDR2 comprising SEQ
ID NO:17; and
[0050] (f) a human light chain variable region CDR3 comprising SEQ
ID NO:21.
[0051] (a) a human heavy chain variable region CDR1 comprising SEQ
ID NO:2;
[0052] (b) a human heavy chain variable region CDR2 comprising SEQ
ID NO:6;
[0053] (c) a human heavy chain variable region CDR3 comprising SEQ
ID NO:10;
[0054] (d) a human light chain variable region CDR1 comprising SEQ
ID NO:14;
[0055] (e) a human light chain variable region CDR2 comprising SEQ
ID NO:18; and
[0056] (f) a human light chain variable region CDR3 comprising SEQ
ID NO:22.
[0057] (a) a human heavy chain variable region CDR1 comprising SEQ
ID NO:3;
[0058] (b) a human heavy chain variable region CDR2 comprising SEQ
ID NO:7;
[0059] (c) a human heavy chain variable region CDR3 comprising SEQ
ID NO:11;
[0060] (d) a human light chain variable region CDR1 comprising SEQ
ID NO:15;
[0061] (e) a human light chain variable region CDR2 comprising SEQ
ID NO:19; and
[0062] (f) a human light chain variable region CDR3 comprising SEQ
ID NO:23.
[0063] (a) a human heavy chain variable region CDR1 comprising SEQ
ID NO:4;
[0064] (b) a human heavy chain variable region CDR2 comprising SEQ
ID NO:8;
[0065] (c) a human heavy chain variable region CDR3 comprising SEQ
ID NO:12;
[0066] (d) a human light chain variable region CDR1 comprising SEQ
ID NO:16;
[0067] (e) a human light chain variable region CDR2 comprising SEQ
ID NO:20; and
[0068] (f) a human light chain variable region CDR3 comprising SEQ
ID NO:24.
[0069] Other preferred antibodies of the invention include isolated
human monoclonal antibodies, or antigen binding portions thereof,
comprising:
[0070] (a) a human heavy chain variable region comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs: 25,
26, 27, and 28; and
[0071] (b) a human light chain variable region comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs: 29,
30, 31, and 32;
[0072] wherein the antibody specifically binds human interferon
alpha receptor 1 with a binding affinity of at least
1.times.10.sup.-8 M or greater affinity.
[0073] Preferred combinations of heavy and light chains include the
following:
[0074] (a) a human heavy chain variable region comprising the amino
acid sequence of SEQ ID NO:25; and
[0075] (b) a human light chain variable region comprising the amino
acid sequence of SEQ ID NO:29.
[0076] (a) a human heavy chain variable region comprising the amino
acid sequence of SEQ ID NO:26; and
[0077] (b) a human light chain variable region comprising the amino
acid sequence of SEQ ID NO:30.
[0078] (a) a human heavy chain variable region comprising the amino
acid sequence of SEQ ID NO:27; and
[0079] (b) a human light chain variable region comprising the amino
acid sequence of SEQ ID NO:31.
[0080] (a) a human heavy chain variable region comprising the amino
acid sequence of SEQ ID NO:28; and
[0081] (b) a human light chain variable region comprising the amino
acid sequence of SEQ ID NO:32.
[0082] Another aspect of the invention pertains to antibodies that
compete for binding to IFNAR-1 with a reference antibody provided
by the invention. Accordingly, in another embodiment, the invention
provides:
[0083] an isolated monoclonal antibody, or antigen binding portion
thereof, wherein the antibody cross-competes for binding to human
interferon alpha receptor 1 with a reference antibody, wherein the
reference antibody is selected from the group consisting of
[0084] a) an antibody comprising a heavy chain variable region
comprising the amino acid sequence of SEQ ID NO: 25; and a light
chain variable region comprising the amino acid sequence of SEQ ID
NO: 29;
[0085] b) an antibody comprising a heavy chain variable region
comprising the amino acid sequence of SEQ ID NO: 26; and a light
chain variable region comprising the amino acid sequence of SEQ ID
NO: 30;
[0086] c) an antibody comprising a heavy chain variable region
comprising the amino acid sequence of SEQ ID NO: 27; and a light
chain variable region comprising the amino acid sequence of SEQ ID
NO: 31; and
[0087] d) an antibody comprising a heavy chain variable region
comprising the amino acid sequence of SEQ ID NO: 28; and a light
chain variable region comprising the amino acid sequence of SEQ ID
NO: 32.
[0088] In certain embodiments, the invention provides a human
antibody, or antigen-binding portion thereof, wherein the antibody
does not bind the same epitope as (i.e., does not cross-compete
with) mouse monoclonal antibody 64G12 (ECACC Deposit No.
92022605).
[0089] The antibodies of the invention can be of any isotype.
Preferred antibodies are of the IgG1, IgG3 or IgG4 isotype. The
antibodies of the invention can be full-length antibodies
comprising variable and constant regions, or they can be
antigen-binding fragments thereof, such as a single chain antibody,
or a Fab or Fab'2 fragment.
[0090] The invention also provides an immunoconjugate comprising an
antibody of the invention, or antigen-binding portion thereof,
linked to a therapeutic agent, such as a cytotoxin or a radioactive
isotope. The invention also provides a bispecific molecule
comprising an antibody, or antigen-binding portion thereof, of the
invention, linked to a second functional moiety having a different
binding specificity than said antibody, or antigen binding portion
thereof.
[0091] Compositions comprising an antibody, or antigen-binding
portion thereof, or immunoconjugate or bispecific molecule of the
invention and a pharmaceutically acceptable carrier are also
provided.
[0092] Nucleic acid molecules encoding the antibodies, or
antigen-binding portions thereof, of the invention are also
encompassed by the invention, as well as expression vectors
comprising such nucleic acids and host cells comprising such
expression vectors. Moreover, the invention provides a transgenic
mouse comprising human immunoglobulin heavy and light chain
transgenes, wherein the mouse expresses an antibody of the
invention, as well as hybridomas prepared from such a mouse,
wherein the hybridoma produces the antibody of the invention.
[0093] The invention also provides methods for making "second
generation" anti-IFNAR-1 antibodies based on the sequences of the
anti-IFNAR-1 antibodies provided herein. For example, the invention
provides a method for preparing an anti-IFNAR-1 antibody
comprising:
[0094] (a) providing: (i) a heavy chain variable region antibody
sequence comprising a CDR1 sequence that is selected from the group
consisting of SEQ ID NOs: 1, 2, 3 and 4, a CDR2 sequence that is
selected from the group consisting of SEQ ID NOs: 5, 6, 7, and 8;
and a CDR3 sequence that is selected from the group consisting of
SEQ ID NOs: 9, 10, 11, and 12; or (ii) a light chain variable
region antibody sequence comprising a CDR1 sequence that is
selected from the group consisting of SEQ ID NOs: 13, 14, 15, and
16, a CDR2 sequence that is selected from the group consisting of
SEQ ID NOs: 17, 18, 19, and 20 and a CDR3 sequence that is selected
from the group consisting of SEQ ID NOs: 21, 22, 23, and 24;
[0095] (b) altering at least one amino acid residue within at least
one variable region antibody sequence, said sequence being selected
from the heavy chain variable region antibody sequence and the
light chain variable region antibody sequence, to create at least
one altered antibody sequence; and
[0096] (c) expressing the altered antibody sequence as a
protein.
[0097] The invention also provides a method for inhibiting
biological activity of a type I interferon on a cell expressing
interferon alpha receptor 1 comprising contacting the cell with the
antibody of the invention, such that the biological activity of the
type I interferon is inhibited. The invention also provides a
method of treating a type I interferon-mediated disease or disorder
in a subject in need of treatment comprising administering to the
subject the antibody, or antigen-binding portion thereof, of the
invention, such that the type-I interferon mediated disease in the
subject is treated. The type I interferon-mediated disease can be,
for example, an interferon alpha-mediated disease.
[0098] Examples of disease or disorders that can be treated using
the methods of the invention include systemic lupus erythematosus,
insulin dependent diabetes mellitus, inflammatory bowel disease,
multiple sclerosis, psoriasis, autoimmune thyroiditis, rheumatoid
arthritis, glomerulonephritis, HIV infection, AIDS, transplant
rejection and graft versus host disease.
[0099] Other features and advantages of the instant invention will
be apparent from the following detailed description and examples
which should not be construed as limiting. The contents of all
references, Genbank entries, patents and published patent
applications cited throughout this application are expressly
incorporated herein by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0100] FIG. 1A shows the nucleotide sequence (SEQ ID NO: 33) and
amino acid sequence (SEQ ID NO: 25) of the heavy chain variable
region of the 3F11 human monoclonal antibody. The CDR1 (SEQ ID NO:
1), CDR2 (SEQ ID NO: 5) and CDR3 (SEQ ID NO: 9) regions are
delineated.
[0101] FIG. 1B shows the nucleotide sequence (SEQ ID NO: 37) and
amino acid sequence (SEQ ID NO: 29) of the light chain variable
region of the 3F11 human monoclonal antibody. The CDR1 (SEQ ID NO:
13), CDR2 (SEQ ID NO: 17) and CDR3 (SEQ ID NO: 21) regions are
delineated.
[0102] FIG. 2A shows the nucleotide sequence (SEQ ID NO: 34) and
amino acid sequence (SEQ ID NO: 26) of the heavy chain variable
region of the 4G5 human monoclonal antibody. The CDR1 (SEQ ID NO:
2), CDR2 (SEQ ID NO: 6) and CDR3 (SEQ ID NO: 10) regions are
delineated.
[0103] FIG. 2B shows the nucleotide sequence (SEQ ID NO: 38) and
amino acid sequence (SEQ ID NO: 30) of the light chain variable
region of the 4G5 human monoclonal antibody. The CDR1 (SEQ ID NO:
14), CDR2 (SEQ ID NO: 18) and CDR3 (SEQ ID NO: 22) regions are
delineated.
[0104] FIG. 3A shows the nucleotide sequence (SEQ ID NO: 35) and
amino acid sequence (SEQ ID NO: 27) of the heavy chain variable
region of the 11E2 human monoclonal antibody. The CDR1 (SEQ ID NO:
3), CDR2 (SEQ ID NO: 7) and CDR3 (SEQ ID NO: 11) regions are
delineated.
[0105] FIG. 3B shows the nucleotide sequence (SEQ ID NO: 39) and
amino acid sequence (SEQ ID NO: 31) of the light chain variable
region of the 11E2 human monoclonal antibody. The CDR1 (SEQ ID NO:
15), CDR2 (SEQ ID NO: 19) and CDR3 (SEQ ID NO: 23) regions are
delineated.
[0106] FIG. 4A shows the nucleotide sequence (SEQ ID NO: 36) and
amino acid sequence (SEQ ID NO: 28) of the heavy chain variable
region of the 9D4 human monoclonal antibody. The CDR1 (SEQ ID NO:
4), CDR2 (SEQ ID NO: 8) and CDR3 (SEQ ID NO: 12) regions are
delineated.
[0107] FIG. 4B shows the nucleotide sequence (SEQ ID NO: 40) and
amino acid sequence (SEQ ID NO: 32) of the light chain variable
region of the 904 human monoclonal antibody. The CDR1 (SEQ ID NO:
16), CDR2 (SEQ ID NO: 20) and CDR3 (SEQ ID NO: 24) regions are
delineated.
[0108] FIG. 5 shows the alignment of the amino acid sequence of the
heavy chain variable region of 3F11 with the human germline V.sub.H
4-34 amino acid sequence (SEQ ID NO: 41).
[0109] FIG. 6 shows the alignment of the amino acid sequence of the
heavy chain variable region of 4G5 with the human germline V.sub.H
4-34 amino acid sequence (SEQ ID NO: 41).
[0110] FIG. 7 shows the alignment of the amino acid sequence of the
heavy chain variable region of 11E2 and 9D4 with the human germline
V.sub.H 5-51 amino acid sequence (SEQ ID NO: 42).
[0111] FIG. 8 shows the alignment of the amino acid sequence of the
light chain variable region of 3F11 with the human germline V.sub.k
L18 amino acid sequence (SEQ ID NO: 43).
[0112] FIG. 9 shows the alignment of the amino acid sequence of the
light chain variable region of 4G5 with the human germline V.sub.k
L18 amino acid sequence (SEQ ID NO: 43).
[0113] FIG. 10 shows the alignment of the amino acid sequence of
the light chain variable region of 11E2 and 9D4 with the human
germline V.sub.k A27 amino acid sequence (SEQ ID NO: 44).
[0114] FIG. 11 is a graph showing the results of experiments
demonstrating that the human monoclonal antibody, 3F11, directed
against human IFNAR-1, does not compete with the mouse monoclonal
antibody 64G12 for binding to IFNAR-1.
DETAILED DESCRIPTION OF THE INVENTION
[0115] The present invention relates to isolated monoclonal
antibodies that bind to Interferon alpha receptor 1 (IFNAR-1) and
that are capable of blocking the action of type I interferons. The
invention provides isolated antibodies, methods of making such
antibodies, immunoconjugates and bispecific molecules comprising
such antibodies and pharmaceutical compositions containing the
antibodies, immunconjugates or bispecific molecules of the
invention. The invention also relates to methods of using the
antibodies to inhibit the binding of a type I interferon to IFNAR-1
on a cell expressing IFNAR-1, for example, in the treatment of
immune mediated disorders, including autoimmune disorders,
transplant rejection and Graft Versus Host Disease (GVHD), in a
subject.
[0116] In order that the present invention may be more readily
understood, certain terms are first defined. Additional definitions
are set forth throughout the detailed description.
[0117] The terms "Interferon alpha receptor-1, " "IFNAR-1," and
"IFNAR-1 antigen" are used interchangeably, and include variants,
isoforms, species homologs of human IFNAR-1, and analogs having at
least one common epitope with IFNAR-1. Accordingly, human
antibodies of the invention may, in certain cases, cross-react with
IFNAR-1 from species other than human, or other proteins which are
structurally related to human IFNAR-1 (e.g., human IFNAR-1
homologs). In other cases, the antibodies may be completely
specific for human IFNAR-1 and not exhibit species or other types
of cross-reactivity.
[0118] The complete cDNA sequence of human IFNAR-1 has the Genbank
accession number NM.sub.--000629.
[0119] The term "type I interferon" as used herein is intended to
refer to members of the type I interferon family of molecules that
are ligands for IFNAR-1 (i.e., members of the type I interferon
family of molecules that are capable of binding IFNAR-1). Examples
of type I interferon ligands are interferon alpha 1, 2a, 2b, 4, 5,
6, 7, 8, 10, 14, 16, 17, 21, interferon beta and interferon
omega.
[0120] The term "immune response" refers to the action of, for
example, lymphocytes, antigen presenting cells, phagocytic cells,
granulocytes, and soluble macromolecules produced by the above
cells or the liver (including antibodies, cytokines, and
complement) that results in selective damage to, destruction of, or
elimination from the human body of invading pathogens, cells or
tissues infected with pathogens, cancerous cells, or, in cases of
autoimmunity or pathological inflammation, normal human cells or
tissues.
[0121] A "signal transduction pathway" refers to the biochemical
relationship between a variety of signal transduction molecules
that play a role in the transmission of a signal from one portion
of a cell to another portion of a cell. As used herein, the phrase
"cell surface receptor" includes, for example, molecules and
complexes of molecules capable of receiving a signal and the
transmission of such a signal across the plasma membrane of a cell.
An example of a "cell surface receptor" of the present invention is
the IFNAR-1 receptor.
[0122] The term "antibody" as referred to herein includes whole
antibodies and any antigen binding fragment (i.e., "antigen-binding
portion") or single chains thereof. An "antibody" refers to a
glycoprotein comprising at least two heavy (H) chains and two light
(L) chains inter-connected by disulfide bonds, or an antigen
binding portion thereof Each heavy chain is comprised of a heavy
chain variable region (abbreviated herein as V.sub.H) and a heavy
chain constant region. The heavy chain constant region is comprised
of three domains, C.sub.H1, C.sub.H2 and C.sub.H3. Each light chain
is comprised of a light chain variable region (abbreviated herein
as V.sub.L) and a light chain constant region. The light chain
constant region is comprised of one domain, C.sub.L. The V.sub.H
and V.sub.L regions can be further subdivided into regions of
hypervariability, termed complementarity determining regions (CDR),
interspersed with regions that are more conserved, termed framework
regions (FR). Each V.sub.H and V.sub.L is composed of three CDRs
and four FRs, arranged from amino-terminus to carboxy-terminus in
the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The
variable regions of the heavy and light chains contain a binding
domain that interacts with an antigen. The constant regions of the
antibodies may mediate the binding of the immunoglobulin to host
tissues or factors, including various cells of the immune system
(e.g., effector cells) and the first component (Clq) of the
classical complement system.
[0123] The term "antigen-binding portion" of an antibody (or simply
"antibody portion"), as used herein, refers to one or more
fragments of an antibody that retain the ability to specifically
bind to an antigen (e.g., IFNAR-1). It has been shown that the
antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Examples of binding fragments
encompassed within the term "antigen-binding portion" of an
antibody include (i) a Fab fragment, a monovalent fragment
consisting of the V.sub.L, V.sub.H, C.sub.L and C.sub.H1 domains;
(ii) a F(ab').sub.2 fragment, a bivalent fragment comprising two
Fab fragments linked by a disulfide bridge at the hinge region;
(iii) a Fd fragment consisting of the V.sub.H and C.sub.H1 domains;
(iv) a Fv fragment consisting of the V.sub.L and V.sub.H domains of
a single arm of an antibody, (v) a dAb fragment (Ward et al.,
(1989) Nature 341:544-546), which consists of a V.sub.H domain; and
(vi) an isolated complementarity determining region (CDR).
Furthermore, although the two domains of the Fv fragment, V.sub.L
and V.sub.H, are coded for by separate genes, they can be joined,
using recombinant methods, by a synthetic linker that enables them
to be made as a single protein chain in which the V.sub.L and
V.sub.H regions pair to form monovalent molecules (known as single
chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426;
and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
Such single chain antibodies are also intended to be encompassed
within the term "antigen-binding portion" of an antibody. These
antibody fragments are obtained using conventional techniques known
to those with skill in the art, and the fragments are screened for
utility in the same manner as are intact antibodies.
[0124] An "isolated antibody", as used herein, is intended to refer
to an antibody that is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
antibody that specifically binds IFNAR-1 is substantially free of
antibodies that specifically bind antigens other than IFNAR-1). An
isolated antibody that specifically binds IFNAR-1 may, however,
have cross-reactivity to other antigens, such as IFNAR-1 molecules
from other species. Moreover, an isolated antibody may be
substantially free of other cellular material and/or chemicals.
[0125] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
composition displays a single binding specificity and affinity for
a particular epitope.
[0126] The term "human antibody", as used herein, is intended to
include antibodies having variable regions in which both the
framework and CDR regions are derived from human germline
immunoglobulin sequences. Furthermore, if the antibody contains a
constant region, the constant region also is derived from human
germline immunoglobulin sequences. The human antibodies of the
invention may include amino acid residues not encoded by human
germline immunoglobulin sequences (e.g., mutations introduced by
random or site-specific mutagenesis in vitro or by somatic mutation
in viva). However, the term "human antibody", as used herein, is
not intended to include antibodies in which CDR sequences derived
from the germline of another mammalian species, such as a mouse,
have been grafted onto human framework sequences.
[0127] The term "human monoclonal antibody" refers to antibodies
displaying a single binding specificity which have variable regions
in which both the framework and CDR regions are derived from human
germline immunoglobulin sequences. In one embodiment, the human
monoclonal antibodies are produced by a hybridoma which includes a
B cell obtained from a transgenic nonhuman animal, e.g., a
transgenic mouse, having a genome comprising a human heavy chain
transgene and a light chain transgene fused to an immortalized
cell.
[0128] The term "recombinant human antibody", as used herein,
includes all human antibodies that are prepared, expressed, created
or isolated by recombinant means, such as (a) antibodies isolated
from an animal (e.g., a mouse) that is transgenic or
transchromosomal for human immunoglobulin genes or a hybridoma
prepared therefrom (described further below), (b) antibodies
isolated from a host cell transformed to express the human
antibody, e.g., from a transfectoma, (c) antibodies isolated from a
recombinant, combinatorial human antibody library, and (d)
antibodies prepared, expressed, created or isolated by any other
means that involve splicing of human immunoglobulin gene sequences
to other DNA sequences. Such recombinant human antibodies have
variable regions in which the framework and CDR regions are derived
from human germline immunoglobulin sequences. In certain
embodiments, however, such recombinant human antibodies can be
subjected to in vitro mutagenesis (or, when an animal transgenic
for human Ig sequences is used, in vivo somatic mutagenesis) and
thus the amino acid sequences of the V.sub.H and V.sub.L regions of
the recombinant antibodies are sequences that, while derived from
and related to human germline V.sub.H and V.sub.L sequences, may
not naturally exist within the human antibody germline repertoire
in vivo.
[0129] As used herein, "isotype" refers to the antibody class
(e.g., IgM or IgG1) that is encoded by the heavy chain constant
region genes.
[0130] As used herein, "specific binding" refers to antibody
binding to a predetermined antigen. Typically, the antibody binds
with a dissociation constant (K.sub.D) of 10.sup.-7 M or less, and
binds to the predetermined antigen with a K.sub.D that is at least
two-fold less than its K.sub.D for binding to a non-specific
antigen (e.g., BSA, casein) other than the predetermined antigen or
a closely-related antigen. The phrases "an antibody recognizing an
antigen" and "an antibody specific for an antigen" are used
interchangeably herein with the term "an antibody which binds
specifically to an antigen".
[0131] The term "K.sub.assoc" or "K.sub.a", as used herein, is
intended to refer to the association rate of a particular
antibody-antigen interaction, whereas the term "K.sub.dis" or
"K.sub.d," as used herein, is intended to refer to the dissociation
rate of a particular antibody-antigen interaction. The term
"K.sub.D", as used herein, is intended to refer to the dissociation
constant, which is obtained from the ratio of K.sub.d to K.sub.a
(i.e,. K.sub.d/K.sub.a) and is expressed as a molar concentration
(M). K.sub.D values for antibodies can be determined using methods
well established in the art. A preferred method for determining the
K.sub.D of an antibody is by using surface plasmon resonance,
preferably using a biosensor system such as a Biacore.RTM.
system.
[0132] As used herein, the term "high affinity" for an IgG antibody
refers to an antibody having a K.sub.D of 10.sup.-8 M or less, more
preferably 10.sup.-9 M or less and even more preferably 10.sup.-10
M or less. However, "high affinity" binding can vary for other
antibody isotypes. For example, "high affinity" binding for an IgM
isotype refers to an antibody having a KD of 10.sup.-7 M or less,
more preferably 10.sup.-8 M or less.
[0133] As used herein, the term "subject" includes any human or
nonhuman animal. The term "nonhuman animal" includes all
vertebrates, e.g., mammals and non-mammals, such as nonhuman
primates, sheep, dogs, cats, horses, cows chickens, amphibians,
reptiles, etc.
[0134] Various aspects of the invention are described in further
detail in the following subsections.
Anti-IFNAR-1 Antibodies
[0135] The antibodies of the invention are characterized by
particular functional features or properties of the antibodies. For
example, the antibodies bind specifically to IFNAR-1, preferably
human IFNAR-1. Additionally, the antibodies may cross react with
IFNAR-1 from one or more non-human primates, such as cynomolgus
monkey and/or rhesus monkey. Preferably, an antibody of the
invention binds to IFNAR-1 with high affinity, for example with a
K.sub.D of 10.sup.-7 M or less, more preferably with a K.sub.D of
10.sup.-8 M or less or 10.sup.-9 M or less or even
5.times.10.sup.-10 M or less or 2.times.10.sup.-10 M or less.
[0136] Furthermore, the antibodies of the invention are capable of
inhibiting the biological activity of type 1 interferons. The
antibodies inhibit the biological activity of at least one type I
interferon, and preferably inhibit the biological activity of
multiple type I interferons (i.e., at least two, more preferably at
least three, or at least four, or at least five, or at least six,
or at least seven, or at least eight, or at least nine, or at least
ten, or at least 11, or at least 12, or at least 13 or at least 14
or at least 15, different subtypes of type I interferon). In a
preferred embodiment, the antibody inhibits the biological activity
of the following type I interferons: .alpha.1, .alpha. 2a, .alpha.
2b, .alpha. 4, .alpha. 5, .alpha. 6, .alpha. 7, .alpha. 8, .alpha.
10, .alpha. 14, .alpha. 16, .alpha. 17, .alpha. 21, beta and omega.
In other preferred embodiments, the antibody inhibits the activity
of lymphoblastoid IFN and/or leukocyte IFN.
[0137] The ability of an antibody to inhibit the biological
activity of type I interferons can be examined in one or more
assays established in the art. Non-limiting examples include
inhibition of Type I IFN-mediated inhibition of Daudi cell
proliferation, inhibition of Type I IFN-induced expression of IP-10
by peripheral blood mononuclear cells (PBMC), inhibition of
dendritic cell development mediated by Systemic Lupus Erythematosus
(SLE) plasma, and inhibition of the anti-viral activity of Type I
IFN. At antibody "inhibits the biological activity of type I
interferons" if it inhibits the activity by at least 20%, more
preferably by at least 30%, even more preferably by at least 40%,
at least 50%, at least 60%, at least 70%, at least 80% or at least
90%, as compared to a non-specific, control antibody.
[0138] In preferred embodiments, the antibody inhibits the activity
of IFN .alpha. 2b in a Daudi cell proliferation assay, inhibits the
activity of IFN omega in a Daudi cell proliferation assay, inhibits
IP-10 secretion by PBMC induced by IFN .alpha. 2b or IFN omega,
and/or inhibits dendritic cell development mediated by SLE
plasma.
[0139] In another preferred embodiment, the antibody does not
cross-compete with (i.e., binds to a different epitope than) the
murine anti-IFNAR-1 antibody 64G12 (deposited as ECACC Deposit No.
92022605).
[0140] Assays to evaluate the functional activities of anti-IFNAR
antibodies are described in further detail in the Examples.
Preferred antibodies of the invention exhibit at least one, more
preferably two, three, four, five or more, of the following
properties:
[0141] a) specifically binds to IFNAR1 (preferably human
IFNAR1);
[0142] b) binds to IFNAR1 with high affinity, such as a K.sub.D of
1.times.10.sup.-8 M or greater affinity;
[0143] c) inhibits the biological activity of multiple Type I
interferons;
[0144] d) inhibits the activity of IFN .alpha. 2b in a Daudi cell
proliferation assay;
[0145] e) inhibits the activity of IFN omega in a Daudi cell
proliferation assay;
[0146] f) inhibits IP-10 secretion by peripheral blood mononuclear
cells induced by IFN .alpha. 2b;
[0147] g) inhibits IP-10 secretion by peripheral blood mononuclear
cells induced by IFN omega;
[0148] h) inhibits dendritic cell development mediated by Systemic
Lupus Erythematosus plasma; and
[0149] i) binds to a different epitope than (i.e., does not
cross-compete with) murine monoclonal antibody 64G12 (ECACC Deposit
No. 92022605).
[0150] Any combination of the above-described functional features,
and/or the functional features as described in the Examples, may be
exhibited by an antibody of the invention.
Monoclonal Antibody 3F11, 4G5, 11E2, and 9D4
[0151] Preferred antibodies of the invention are the human
monoclonal antibodies 3F11, 4G5, 11E2, and 9D4, isolated and
structurally characterized as described in the Examples. The
V.sub.H amino acid sequences of 3F11, 4G5, 11E2, and 9D4 are shown
in SEQ ID NOs: 25, 26, 27, and 28, respectively. The V.sub.L amino
acid sequences of 3F11, 4G5, 11E2, and 9D4 are shown in SEQ ID NOs:
29, 30, 31, and 32, respectively.
[0152] Given that each of these antibodies can bind to IFNAR-1, the
V.sub.H and V.sub.L sequences can be "mixed and matched" to create
other anti-IFNAR-1 binding molecules of the invention. IFNAR-1
binding of such "mixed and matched" antibodies can be tested using
the binding assays described herein (e.g., ELISAs) and/or using the
type I IFN functional inhibition assays described in the Examples.
Preferably, when V.sub.H and V.sub.L chains are mixed and matched,
a V.sub.H sequence from a particular V.sub.H/V.sub.L pairing is
replaced with a structurally similar V.sub.H sequence. Likewise,
preferably a V.sub.L sequence from a particular V.sub.H/V.sub.L
pairing is replaced with a structurally similar V.sub.L sequence.
For example, the V.sub.H and V.sub.L sequences of 3F11 and 4G5 are
particularly amenable for mixing and matching, since these
antibodies use V.sub.H and V.sub.L sequences derived from the same
germline sequences (V.sub.H 4-34 and V.sub.k L18) and thus they
exhibit structural similarity. In addition, the V.sub.H and V.sub.L
sequences of 11E2 and 9D4 are particularly amenable for mixing and
matching, since these antibodies use V.sub.H and V.sub.L sequences
derived from the same germline sequences (V.sub.H 5-51 and V.sub.k
A27) and thus they exhibit structural similarity.
[0153] Accordingly, in one aspect, the invention provides an
isolated monoclonal antibody, or antigen binding portion thereof,
comprising:
[0154] (a) a heavy chain variable region comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs: 25, 26,
27, and 28; and
[0155] (b) a light chain variable region comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs: 29, 30,
31, and 32;
[0156] wherein the antibody specifically binds IFNAR-1.
Preferred heavy and light chain combinations include:
[0157] (a) a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 25; and (b) a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 29; or
[0158] (a) a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 26; and (b) a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 30; or
[0159] (a) a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 27; and (b) a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 31; or
[0160] (a) a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 28; and (b) a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 32.
[0161] In another aspect, the invention provides antibodies that
comprise the heavy chain and light chain CDR1s, CDR2s and CDR3s of
3F11, 4G5, 11E2, and 9D4, or combinations thereof. The amino acid
sequences of the V.sub.H CDR1s of 3F11, 4G5, 11E2, and 9D4 are
shown in SEQ ID NOs: 1, 2, 3, and 4. The amino acid sequences of
the V.sub.H CDR2s of 3F11, 4G5, 11E2, and 9D4 are shown in SEQ ID
NOs: 5, 6, 7, and 8. The amino acid sequences of the V.sub.H CDR3s
of 3F11, 4G5, 11E2, and 9D4 are shown in SEQ ID NOs: 9, 10, 11, and
12. The amino acid sequences of the V.sub.k CDR1s of 3F11, 4G5,
11E2, and 9D4 are shown in SEQ ID NOs: 13, 14, 15, and 16. The
amino acid sequences of the V.sub.k CDR2s of 3F11, 4G5, 11E2, and
9D4 are shown in SEQ ID NOs: 17, 18, 19, and 20. The amino acid
sequences of the V.sub.k CDR3s of 3F11, 4G5, 11E2, and 9D4 are
shown in SEQ ID NOs: 21, 22, 23, and 24. The CDR regions are
delineated using the Kabat system (Kabat, E. A., et al. (1991)
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No.
91-3242).
[0162] Given that each of these antibodies can bind to IFNAR-1 and
that antigen-binding specificity is provided primarily by the CDR1,
2 and 3 regions, the V.sub.H CDR1, 2 and 3 sequences and V.sub.k
CDR1, 2 and 3 sequences can be "mixed and matched" (i.e., CDRs from
different antibodies can be mixed and match, although each antibody
must contain a V.sub.H CDR1, 2 and 3 and a V.sub.k CDR1, 2 and 3)
to create other anti-IFNAR-1 binding molecules of the invention.
IFNAR-1 binding of such "mixed and matched" antibodies can be
tested using the binding assays described above and in the Examples
(e.g., ELISAs). Preferably, when V.sub.H CDR sequences are mixed
and matched, the CDR1, CDR2 and/or CDR3 sequence from a particular
V.sub.H sequence is replaced with a structurally similar CDR
sequence(s). Likewise, when V.sub.k CDR sequences are mixed and
matched, the CDR1, CDR2 and/or CDR3 sequence from a particular
V.sub.k sequence preferably is replaced with a structurally similar
CDR sequence(s). For example, the V.sub.H CDR1s of 3F11 and 4G5
share some structural similarity and therefore are amenable to
mixing and matching. As another example, the V.sub.H CDR1s of 11E2
and 9D4 share some structural similarity and therefore are amenable
to mixing and matching. As yet another example, the V.sub.K CDR1s
of 3F11 and 4G5 share some structural similarity. As yet another
example, the V.sub.H CDR1s of 11E2 and 9D4 share some structural
similarity. It will be readily apparent to the ordinarily skilled
artisan that novel V.sub.H and V.sub.L sequences can be created by
substituting one or more V.sub.H and/or V.sub.L CDR region
sequences with structurally similar sequences from the CDR
sequences disclosed herein for monoclonal antibodies antibodies
3F11, 4G5, 11E2, and 9D4.
[0163] Accordingly, in another aspect, the invention provides an
isolated monoclonal antibody, or antigen binding portion thereof
comprising:
[0164] (a) a heavy chain variable region CDR1 comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs: 1,
2, 3, and 4;
[0165] (b) a heavy chain variable region CDR2 comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs: 5,
6, 7, and 8;
[0166] (c) a heavy chain variable region CDR3 comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs: 9,
10, 11, and 12;
[0167] (d) a light chain variable region CDRI comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs: 13,
14, 15, and 16;
[0168] (e) a light chain variable region CDR2 comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs: 17,
18, 19, and 20; and
[0169] (f) a light chain variable region CDR3 comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs: 21,
22, 23, and 24;
[0170] wherein the antibody specifically binds IFNAR-1.
In a preferred embodiment, the antibody comprises:
[0171] (a) a heavy chain variable region CDR1 comprising SEQ ID NO:
1;
[0172] (b) a heavy chain variable region CDR2 comprising SEQ ID NO:
5;
[0173] (c) a heavy chain variable region CDR3 comprising SEQ ID NO:
9;
[0174] (d) a light chain variable region CDR1 comprising SEQ ID NO:
13;
[0175] (e) a light chain variable region CDR2 comprising SEQ ID NO:
17; and
[0176] (f) a light chain variable region CDR3 comprising SEQ ID NO:
21.
In another preferred embodiment, the antibody comprises:
[0177] (a) a heavy chain variable region CDR1 comprising SEQ ID NO:
2;
[0178] (b) a heavy chain variable region CDR2 comprising SEQ ID NO:
6;
[0179] (c) a heavy chain variable region CDR3 comprising SEQ ID NO:
10;
[0180] (d) a light chain variable region CDR1 comprising SEQ ID NO:
14;
[0181] (e) a light chain variable region CDR2 comprising SEQ ID NO:
18; and
[0182] (f) a light chain variable region CDR3 comprising SEQ ID NO:
22.
In another preferred embodiment, the antibody comprises:
[0183] (a) a heavy chain variable region CDR1 comprising SEQ ID NO:
3;
[0184] (b) a heavy chain variable region CDR2 comprising SEQ ID NO:
7;
[0185] (c) a heavy chain variable region CDR3 comprising SEQ ID NO:
11;
[0186] (d) a light chain variable region CDR1 comprising SEQ ID NO:
15;
[0187] (e) a light chain variable region CDR2 comprising SEQ ID NO:
19; and
[0188] (f) a light chain variable region CDR3 comprising SEQ ID NO:
23.
In another preferred embodiment, the antibody comprises:
[0189] (a) a heavy chain variable region CDR1 comprising SEQ ID NO:
4;
[0190] (b) a heavy chain variable region CDR2 comprising SEQ ID NO:
8;
[0191] (c) a heavy chain variable region CDR3 comprising SEQ ID NO:
12;
[0192] (d) a light chain variable region CDR1 comprising SEQ ID NO:
16;
[0193] (e) a light chain variable region CDR2 comprising SEQ ID NO:
20; and
[0194] (f) a light chain variable region CDR3 comprising SEQ ID NO:
24.
Antibodies that Bind to the Same Epitope as 3F11, 4G5, 11E2, and
9D4
[0195] In another embodiment, the invention provides antibodies
that bind to the same epitope on human IFNAR-1 as the monoclonal
antibodies 3F11, 4G5, 11E2, or 9D4 (having V.sub.H sequences as
shown in SEQ ID NOs: 25, 26, 27, and 28, respectively, and V.sub.L
sequences as shown in SEQ ID NOs: 29, 30, 31, 32, respectively).
Such antibodies can be identified based on their ability to
cross-compete with 3F11, 4G5, 11E2, or 9D4 in standard IFNAR-1
binding assays. The ability of a test antibody to inhibit the
binding of 3F11, 4G5, 11E2, or 9D4 to human IFNAR-1 demonstrates
that the test antibody can compete with 3F11, 4G5, 11E2, or 9D4 for
binding to human IFNAR-1 and thus binds to the same epitope on
human IFNAR-1 as 3F11, 4G5, 11E2, or 9D4. In a preferred
embodiment, the antibody that binds to the same epitope on human
1FNAR-1 as 3F11, 4G5, 11E2, or 9D4 is a human monoclonal antibody.
Such human monoclonal antibodies can be prepared and isolated as
described in the Examples.
[0196] In another preferred embodiment, the antibody binds to a
different epitope than (i.e., does not cross-compete with) the
mouse monoclonal antibody 64G12 (ECACC Deposit No. 92022605).
Antibodies Having Particular Germline Sequences
[0197] In certain embodiments, an antibody of the invention
comprises a heavy chain variable region from a particular germline
heavy chain immunoglobulin gene and/or a light chain variable
region from a particular germline light chain immunoglobulin
gene.
[0198] For example, in a preferred embodiment, the invention
provides an isolated anti-IFNAR-1 monoclonal antibody, or an
antigen-binding portion thereof, wherein the antibody:
[0199] (a) comprises a heavy chain variable region of a human VH
4-34 or 5-51 gene;
[0200] (b) comprises a light chain variable region of a human Vk
L18 or A27 gene; and
[0201] (c) the antibody specifically binds to IFNAR-1.
[0202] Examples of antibodies having V.sub.H and V.sub.K of VH 4-34
and Vk L18, respectively, include 3F11 and 4G5. Examples of
antibodies having V.sub.H and V.sub.K of VH 5-51 and Vk A27,
respectively, include 11E2 and 9D4.
[0203] As used herein, a human antibody comprises heavy or light
chain variable regions"of' or "derived from" or "the product or a
particular germline sequence if the variable regions of the
antibody are obtained from a system that uses human germline
immunoglobulin genes. Such systems include immunizing a transgenic
mouse carrying human immunoglobulin genes with the antigen of
interest or screening a human immunoglobulin gene library displayed
on phage with the antigen of interest. A human antibody that is
"of' or "derived from" or "the product of a human germline
immunoglobulin sequence can be identified as such by comparing the
amino acid sequence of the human antibody to the amino acid
sequences of human germline immunoglobulins and selecting the human
germline immunoglobulin sequence that is closest in sequence (i.e.,
greatest % identity) to the sequence of the human antibody. A human
antibody that is "of' or "derived from" or "the product of a
particular human germline immunoglobulin sequence may contain amino
acid differences as compared to the germline sequence, due to, for
example, naturally-occurring somatic mutations or intentional
introduction of site-directed mutation. However, a selected human
antibody typically is at least 90% identical in amino acids
sequence to an amino acid sequence encoded by a human germline
immunoglobulin gene and contains amino acid residues that identify
the human antibody as being human when compared to the germline
immunoglobulin amino acid sequences of other species (e.g., murine
germline sequences). In certain cases, a human antibody may be at
least 95%, or even at least 96%, 97%, 98%, or 99% identical in
amino acid sequence to the amino acid sequence encoded by the
germline immunoglobulin gene. Typically, a human antibody derived
from a particular human germline sequence will display no more than
10 amino acid differences from the amino acid sequence encoded by
the human germline immunoglobulin gene. In certain cases, the human
antibody may display no more than 5, or even no more than 4, 3, 2,
or 1 amino acid difference from the amino acid sequence encoded by
the germline immunoglobulin gene.
Homologous Antibodies
[0204] In yet another embodiment, an antibody of the invention
comprises heavy and light chain variable regions comprising amino
acid sequences that are homologous to the amino acid sequences of
the preferred antibodies described herein, and wherein the
antibodies retain the desired functional properties of the
anti-IFNAR-1 antibodies of the invention. For example, the
invention provides an isolated monoclonal antibody, or antigen
binding portion thereof, comprising a heavy chain variable region
and a light chain variable region, wherein:
[0205] (a) the heavy chain variable region comprises an amino acid
sequence that is at least 80% homologous to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 25, 26, 27, and
28;
[0206] (b) the light chain variable region comprises an amino acid
sequence that is at least 80% homologous to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 29, 30, 31, and
32;
[0207] (c) the antibody specifically binds to IFNAR-1 and exhibits
at least one of the functional properties described herein,
preferably several of the functional properties described
herein.
[0208] In other embodiments, the V.sub.H and/or V.sub.L amino acid
sequences may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to
the sequences set forth above. An antibody having V.sub.H and
V.sub.L regions having high (i.e., 80% or greater) homology to the
V.sub.H and V.sub.L regions of the sequences set forth above, can
be obtained by mutagenesis (e.g., site-directed or PCR-mediated
mutagenesis) of nucleic acid molecules encoding SEQ ID NOs: 33, 34,
35, 36, 37, 38, 39, or 40, followed by testing of the encoded
altered antibody for retained function (i.e., the functions set
forth in (c), (d) and (e) above) using the functional assays
described herein.
[0209] As used herein, the percent homology between two amino acid
sequences is equivalent to the percent identity between the two
sequences. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., % homology=# of identical positions/total # of
positions.times.100), taking into account the number of gaps, and
the length of each gap, which need to be introduced for optimal
alignment of the two sequences. The comparison of sequences and
determination of percent identity between two sequences can be
accomplished using a mathematical algorithm, as described in the
non-limiting examples below.
[0210] The percent identity between two amino acid sequences can be
determined using the algorithm of E. Meyers and W. Miller (Comput.
Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the
ALIGN program (version 2.0), using a PAM120 weight residue table, a
gap length penalty of 12 and a gap penalty of 4. In addition, the
percent identity between two amino acid sequences can be determined
using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970))
algorithm which has been incorporated into the GAP program in the
GCG software package (available at http://www.gcg.com), using
either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of
16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or
6.
[0211] Additionally or alternatively, the protein sequences of the
present invention can further be used as a "query sequence" to
perform a search against public databases to, for example, identify
related sequences. Such searches can be performed using the XBLAST
program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.
215:403-10. BLAST protein searches can be performed with the BLAST
program, score=50, wordlength=3 to obtain amino acid sequences
homologous to the antibody molecules of the invention. To obtain
gapped alignments for comparison purposes, Gapped BLAST can be
utilized as described in Altschul et al., (1997) Nucleic Acids Res.
25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs,
the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
Antibodies with Conservative Modifications
[0212] In certain embodiments, an antibody of the invention
comprises a heavy chain variable region comprising CDR1, CDR2 and
CDR3 sequences and a light chain variable region comprising CDR1,
CDR2 and CDR3 sequences, wherein one or more of these CDR sequences
comprise specified amino acid sequences based on the preferred
antibodies described herein (e.g., 3F11, 4G5, 11E2, and 9D4), or
conservative modifications thereof, and wherein the antibodies
retain the desired functional properties of the anti-IFNAR-1
antibodies of the invention. Accordingly, the invention provides an
isolated monoclonal antibody, or antigen binding portion thereof,
comprising a heavy chain variable region comprising CDR1, CDR2, and
CDR3 sequences and a light chain variable region comprising CDR1,
CDR2, and CDR3 sequences, wherein:
[0213] (a) the heavy chain variable region CDR3 sequence comprises
an amino acid sequence selected from the group consisting of amino
acid sequences of SEQ ID NOs: 9, 10, 11, and 12, and conservative
modifications thereof;
[0214] (b) the light chain variable region CDR3 sequence comprises
an amino acid sequence selected from the group consisting of amino
acid sequences of SEQ ID NOs: 21, 22, 23, and 24, and conservative
modifications thereof; and
[0215] (c) the antibody specifically binds to IFNAR-1 and exhibits
at least one of the functional properties described herein, more
preferably several of the functional properties described
herein.
[0216] In a further embodiment, the heavy chain variable region
CDR2 sequence comprises an amino acid sequence selected from the
group consisting of amino acid sequences of SEQ ID NOs: 5, 6, 7,
and 8, and conservative modifications thereof; and the light chain
variable region CDR2 sequence comprises an amino acid sequence
selected from the group consisting of amino acid sequences of SEQ
ID NOs: 17, 18, 19, and 20, and conservative modifications thereof.
In a still further embodiment, the heavy chain variable region CDR1
sequence comprises an amino acid sequence selected from the group
consisting of amino acid sequences of SEQ ID NOs: 1, 2, 3, and 4,
and conservative modifications thereof; and the light chain
variable region CDR1 sequence comprises an amino acid sequence
selected from the group consisting of amino acid sequences of SEQ
ID NOs: 13, 14, 15, and 16, and conservative modifications
thereof.
[0217] As used herein, the term "conservative sequence
modifications" is intended to refer to amino acid modifications
that do not significantly affect or alter the binding
characteristics of the antibody containing the amino acid sequence.
Such conservative modifications include amino acid substitutions,
additions and deletions. Modifications can be introduced into an
antibody of the invention by standard techniques known in the art,
such as site-directed mutagenesis and PCR-mediated mutagenesis.
Conservative amino acid substitutions are ones in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine, tryptophan),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one
or more amino acid residues within the CDR regions of an antibody
of the invention can be replaced with other amino acid residues
from the same side chain family and the altered antibody can be
tested for retained function (i.e., the functions set forth in (c),
(d) and (e) above) using the functional assays described
herein.
Engineered and Modified Antibodies
[0218] An antibody of the invention further can be prepared using
an antibody having one or more of the V.sub.H and/or V.sub.L
sequences disclosed herein as starting material to engineer a
modified antibody, which modified antibody may have altered
properties from the starting antibody. An antibody can be
engineered by modifying one or more residues within one or both
variable regions (i.e., V.sub.H and/or V.sub.L), for example within
one or more CDR regions and/or within one or more framework
regions. Additionally or alternatively, an antibody can be
engineered by modifying residues within the constant region(s), for
example to alter the effector function(s) of the antibody.
[0219] One type of variable region engineering that can be
performed is CDR grafting. Antibodies interact with target antigens
predominantly through amino acid residues that are located in the
six heavy and light chain complementarity determining regions
(CDRs). For this reason, the amino acid sequences within CDRs are
more diverse between individual antibodies than sequences outside
of CDRs. Because CDR sequences are responsible for most
antibody-antigen interactions, it is possible to express
recombinant antibodies that mimic the properties of specific
naturally occurring antibodies by constructing expression vectors
that include CDR sequences from the specific naturally occurring
antibody grafted onto framework sequeikes from a different antibody
with different properties (see, e.g., Riechmann, L. et al. (1998)
Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525;
Queen, C. et al. (1989) Proc. Natl. Acad. See. U.S.A.
86:10029-10033; U.S. Pat. No. 5,225,539 to Winter; and U.S. Pat.
Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et
al.)
[0220] Accordingly, another embodiment of the invention pertains to
an isolated monoclonal antibody, or antigen binding portion
thereof, comprising: a heavy chain variable region comprising CDR1,
CDR2, and CDR3 sequences comprising an amino acid sequence selected
from the group consisting of SEQ ID NOs: 1, 2, 3, and 4, SEQ ID
NOs: 5, 6, 7, and 8 and SEQ ID NOs: 9, 10, 11, and 12,
respectively, and a light chain variable region comprising CDR1,
CDR2, and CDR3 sequences comprising an amino acid sequence selected
from the group consisting of SEQ ID NOs: 13, 14, 15, and 16, SEQ ID
NOs: 17, 18, 19, and 20 and SEQ ID NOs: 21, 22, 23, and 24,
respectively. Thus, such antibodies contain the V.sub.H and V.sub.L
CDR sequences of monoclonal antibodies 3F11, 4G5, 11E2, or 9D4 yet
may contain different framework sequences from these
antibodies.
[0221] Such framework sequences can be obtained from public DNA
databases or published references that include germline antibody
gene sequences. For example, germline DNA sequences for human heavy
and light chain variable region genes can be found in the "VBase"
human germline sequence database (available on the Internet at
www.mrc-cpe.cam.ac.uk/vbase), as well as in Kabat, E. A., et al.
(1991) Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242; Tomlinson, I. M., et al. (1992) "The
Repertoire of Human Germline V.sub.H Sequences Reveals about Fifty
Groups of V.sub.H Segments with Different Hypervariable Loops" J.
Mol. Biol. 227:776-798; and Cox, J. P. L. et al. (1994) "A
Directory of Human Germ-line V.sub.H Segments Reveals a Strong Bias
in their Usage" Eur. J. Immunol. 24:827-836; the contents of each
of which are expressly incorporated herein by reference.
[0222] Preferred framework sequences for use in the antibodies of
the invention are those that are structurally similar to the
framework sequences used by selected antibodies of the invention,
e.g., similar to the V.sub.H 4-34 and V.sub.L L18 framework
sequences used by the 3F11 and 4G5 monoclonal antibodies, or the
V.sub.H 5-51 and V.sub.L A27 framework sequences used by the 11E2
and 9D4 monoclonal antibodies. The V.sub.H CDR1, 2 and 3 sequences
of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, and the
V.sub.L CDR1, 2 and 3 sequences of SEQ ID NOs: 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, and 24 can be grafted onto framework
regions that have the same sequence as that found in the germline
immunoglobulin gene from which the framework sequence derive, or
the CDR sequences can be grafted onto framework regions that
contain one or more mutations as compared to the germline
sequences. For example, it has been found that in certain instances
it is beneficial to mutate residues within the framework regions to
maintain or enhance the antigen binding ability of the antibody
(see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and
6,180,370 to Queen et al).
[0223] Another type of variable region modification is to mutate
amino acid residues within the V.sub.H and/or V.sub.L CDR1, CDR2
and/or CDR3 regions to thereby improve one or more binding
properties (e.g., affinity) of the antibody of interest.
Site-directed mutagenesis or PCR-mediated mutagenesis can be
performed to introduce the mutations) and the effect on antibody
binding, or other functional property of interest, can be evaluated
in in vitro or in vivo assays as described herein and provided in
the Examples. Preferably conservative modifications (as discussed
above) are introduced. The mutations may be amino acid
substitutions, additions or deletions, but are preferably
substitutions. Moreover, typically no more than five residues are
altered within a CDR region are altered.
[0224] Accordingly, in another embodiment, the invention provides
isolated anti-IFNAR-1 monoclonal antibodies, or antigen binding
portions thereof, comprising a heavy chain variable region
comprising: (a) a V.sub.H CDR1 region comprising an amino acid
sequence selected from the group consisting of SEQ ID NO: 1, 2, 3,
and 4, or an amino acid sequence having one, two, three, four or
five amino acid substitutions, deletions or additions as compared
to an amino acid sequence selected from the group consisting of SEQ
ID NO: 1, 2, 3, and 4; (b) a V.sub.H CDR2 region comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO: 5, 6, 7, and 8, or an amino acid sequence having one, two,
three, four or five amino acid substitutions, deletions or
additions as compared to an amino acid sequence selected from the
group consisting of SEQ ID NO: 5, 6, 7, and 8; (c) a V.sub.H CDR3
region comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 9, 10, 11, and 12, or an amino acid
sequence having one, two, three, four or five amino acid
substitutions, deletions or additions as compared to an amino acid
sequence selected from the group consisting of SEQ ID NO: 9, 10,
11, and 12; (d) a V.sub.L CDR1 region comprising an amino acid
sequence selected from the group consisting of SEQ ID NO: 13, 14,
15, and 16, or an amino acid sequence having one, two, three, four
or five amino acid substitutions, deletions or additions as
compared to an amino acid sequence selected from the group
consisting of SEQ ID NO: 13, 14, 15, and 16; (e) a V.sub.L CDR2
region comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 17, 18, 19, and 20, or an amino acid
sequence having one, two, three, four or five amino acid
substitutions, deletions or additions as compared to an amino acid
sequence selected from the group consisting of SEQ ID NO: 17, 18,
19, and 20; and (f) a V.sub.L CDR3 region comprising an amino acid
sequence selected from the group consisting of SEQ ID NO: 21, 22,
23, and 24, or an amino acid sequence having one, two, three, four
or five amino acid substitutions, deletions or additions as
compared to an amino acid sequence selected from the group
consisting of SEQ ID NO: 17, 18, 19, and 20.
[0225] Engineered antibodies of the invention include those in
which modifications have been made to framework residues within
V.sub.H and/or V.sub.L, e.g. to improve the properties of the
antibody. Typically such framework modifications are made to
decrease the immunogenicity of the antibody. For example, one
approach is to "backmutate" one or more framework residues to the
corresponding germline sequence. More specifically, an antibody
that has undergone somatic mutation may contain framework residues
that differ from the germline sequence from which the antibody is
derived. Such residues can be identified by comparing the antibody
framework sequences to the germline sequences from which the
antibody is derived. For example, for 3F11, amino acid residue #43
(within FR2) of V.sub.H is a threonine whereas this residue in the
corresponding V.sub.H 4-34 germline sequence is an alanine (see
FIG. 5). To return the framework region sequences to their germline
configuration, the somatic mutations can be "backmutated" to the
germline sequence by, for example, site-directed mutagenesis or
PCR-mediated mutagenesis (e.g., residue 43 of the V.sub.H of 3F11
can be "backmutated" from threonine to alanine). As another
example, for 4G5, amino acid residue #81 (within FR3) of V.sub.H is
an asparagine whereas this residue in the corresponding V.sub.H
4-34 germline sequence is a lysine (see FIG. 6). To return the
framework region sequences to their germline configuration, the
somatic mutations can be "backmutated" to from asparagine to
lysine. As another example, for 11E2 and 9D4, amino acid residue
#28 (within FR1) of V.sub.H is an isoleucine whereas this residue
in the corresponding V.sub.H 5-51 germline sequence is a serine
(see FIG. 7). To return the framework region sequences to their
germline configuration, the somatic mutations can be "backmutated"
to from isoleucine to serine. Such "backmutated" antibodies are
also intended to be encompassed by the invention.
[0226] Another type of framework modification involves mutating one
or more residues within the framework region, or even within one or
more CDR regions, to remove T cell epitopes to thereby reduce the
potential immunogenicity of the antibody. This approach is also
referred to as "deimmunization" and is described in further detail
in U.S. Patent Publication No. 20030153043 by Carr et al.
[0227] In addition or alternative to modifications made within the
framework or CDR regions, antibodies of the invention may be
engineered to include modifications within the Fc region, typically
to alter one or more functional properties of the antibody, such as
serum half-life, complement fixation, Fc receptor binding, and/or
antigen-dependent cellular cytotoxicity. Furthermore, an antibody
of the invention may be chemically modified (e.g., one or more
chemical moieties can be attached to the antibody) or be modified
to alter it's glycosylation, again to alter one or more functional
properties of the antibody. Each of these embodiments is described
in further detail below. The numbering of residues in the Fc region
is that of the EU index of Kabat.
[0228] In one embodiment, the hinge region of CH1 is modified such
that the number of cysteine residues in the hinge region is
altered, e.g., increased or decreased. This approach is described
further in U.S. Pat. No. 5,677,425 by Bodmer et al. The number of
cysteine residues in the hinge region of CH1 is altered to, for
example, facilitate assembly of the light and heavy chains or to
increase or decrease the stability of the antibody.
[0229] In another embodiment, the Fc hinge region of an antibody is
mutated to decrease the biological half life of the antibody. More
specifically, one or more amino acid mutations are introduced into
the CH2-CH3 domain interface region of the Fc-hinge fragment such
that the antibody has impaired Staphylococcyl protein A (SpA)
binding relative to native Fc-hinge domain SpA binding. This
approach is described in further detail in U.S. Pat. No. 6,165,745
by Ward et al.
[0230] In another embodiment, the antibody is modified to increase
its biological half life. Various approaches are possible. For
example, one or more of the following mutations can be introduced:
T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375 to
Ward. Alternatively, to increase the biological half life, the
antibody can be altered within the CH1 or CL region to contain a
salvage receptor binding epitope taken from two loops of a CH2
domain of an Fc region of an IgG, as described in U.S. Pat. Nos.
5,869,046 and 6,121,022 by Presta et al.
[0231] In yet other embodiments, the Fc region is altered by
replacing at least one amino acid residue with a different amino
acid residue to alter the effector function(s) of the antibody. For
example, one or more amino acids selected from amino acid residues
234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a
different amino acid residue such that the antibody has an altered
affinity for an effector ligand but retains the antigen-binding
ability of the parent antibody. The effector ligand to which
affinity is altered can be, for example, an Fc receptor or the C1
component of complement. This approach is described in further
detail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et
al.
[0232] In another example, one or more amino acids selected from
amino acid residues 329, 331 and 322 can be replaced with a
different amino acid residue such that the antibody has altered C1q
binding and/or reduced or abolished complement dependent
cytotoxicity (CDC). This approach is described in further detail in
U.S. Pat. Nos. 6,194,551 by Idusogie et al.
[0233] In another example, one or more amino acid residues within
amino acid positions 231 and 239 are altered to thereby alter the
ability of the antibody to fix complement. This approach is
described further in PCT Publication WO 94/29351 by Bodmer et
al.
[0234] In yet another example, the Fc region is modified to
increase the ability of the antibody to mediate antibody dependent
cellular cytotoxicity (ADCC) and/or to increase the affinity of the
antibody for an Fc.gamma. receptor by modifying one or more amino
acids at the following positions: 238, 239, 248, 249, 252, 254,
255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283,
285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305,
307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333,
334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398,
414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is
described further in PCT Publication WO 00/42072 by Presta.
Moreover, the binding sites on human IgG1 for Fc.gamma.R1,
Fc.gamma.RII, Fc.gamma.RIII and FcRn have been mapped and variants
with improved binding have been described (see Shields, R. L. et
al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at
positions 256, 290, 298, 333, 334 and 339 were shown to improve
binding to Fc.gamma.RIII. Additionally, the following combination
mutants were shown to improve Fc.gamma.RIII binding: T256A/S298A,
S298A/E333A, S298A/K224A and S298A/E333A/K334A.
[0235] In still another embodiment, the glycosylation of an
antibody is modified. For example, an aglycoslated antibody can be
made (i.e., the antibody lacks glycosylation). Glycosylation can be
altered to, for example, increase the affinity of the antibody for
antigen. Such carbohydrate modifications can be accomplished by,
for example, altering one or more sites of glycosylation within the
antibody sequence. For example, one or more amino acid
substitutions can be made that result in elimination of one or more
variable region framework glycosylation sites to thereby eliminate
glycosylation at that site. Such aglycosylation may increase the
affinity of the antibody for antigen. Such an approach is described
in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 by Co
et al.
[0236] Additionally or alternatively, an antibody can be made that
has an altered type of glycosylation, such as a hypofucosylated
antibody having reduced amounts of fucosyl residues or an antibody
having increased bisecting GlcNac structures. Such altered
glycosylation patterns have been demonstrated to increase the ADCC
ability of antibodies. Such carbohydrate modifications can be
accomplished by, for example, expressing the antibody in a host
cell with altered glycosylation machinery. Cells with altered
glycosylation machinery have been described in the art and can be
used as host cells in which to express recombinant antibodies of
the invention to thereby produce an antibody with altered
glycosylation. For example, EP 1,176,195 by Hanai et al. describes
a cell line with a functionally disrupted FUT8 gene, which encodes
a fucosyl transferase, such that antibodies expressed in such a
cell line exhibit hypofucosylation. PCT Publication WO 03/035835 by
Presta describes a variant CHO cell line, Lec13 cells, with reduced
ability to attach fucose to Asn(297)-linked carbohydrates, also
resulting in hypofucosylation of antibodies expressed in that host
cell (see also Shields, R. L. et al. (2002) J. Biol. Chem.
277:26733-26740). PCT Publication WO 99/54342 by Umana et al.
describes cell lines engineered to express glycoprotein-modifying
glycosyl transferases (e.g.,
beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that
antibodies expressed in the engineered cell lines exhibit increased
bisecting GlcNac structures which results in increased ADCC
activity of the antibodies (see also Umana et al. (1999) Nat.
Biotech. 17:176-180).
[0237] Another modification of the antibodies herein that is
contemplated by the invention is pegylation. An antibody can be
pegylated to, for example, increase the biological (e.g., serum)
half life of the antibody. To pegylate an antibody, the antibody,
or fragment thereof, typically is reacted with polyethylene glycol
(PEG), such as a reactive ester or aldehyde derivative of PEG,
under conditions in which one or more PEG groups become attached to
the antibody or antibody fragment. Preferably, the pegylation is
carried out via an acylation reaction or an alkylation reaction
with a reactive PEG molecule (or an analogous reactive
water-soluble polymer). As used herein, the term "polyethylene
glycol" is intended to encompass any of the forms of PEG that have
been used to derivative other proteins, such as mono (C1-C10)
alkoxy- or aryloxy-polyethylene glycol or polyethylene
glycol-maleimide. In certain embodiments, the antibody to be
pegylated is an aglycosylated antibody. Methods for pegylating
proteins are known in the art and can be applied to the antibodies
of the invention. See for example, EP 0 154 316 by Nishimura et al.
and EP 0 401 384 by Ishikawa et al.
Methods of Engineering Antibodies
[0238] Thus, in another aspect of the invention, the structural
features of anti-IFNAR-1 antibodies of the invention, e.g. 3F11,
4G5, 11E2, and 9D4 are used to create structurally related
anti-IFNAR-1 antibodies that retain at least one functional
property of the antibodies of the invention, such as binding to
IFNAR-1. For example, one or more CDR regions of 3F11, 4G5, 11E2,
or 9D4, or mutations thereof, can be combined recombinantly with
known framework regions and/or other CDRs to create additional,
recombinantly-engineered, anti-IFNAR-1 antibodies of the invention,
as discussed above. Other types of modifications include those
described in the previous section. The starting material for the
engineering method is one or more of the V.sub.H and/or V.sub.L
sequences provided herein, or one or more CDR regions thereof. To
create the engineered antibody, it is not necessary to actually
prepare (i.e., express as a protein) an antibody having one or more
of the V.sub.H and/or V.sub.L sequences provided herein, or one or
more CDR regions thereof. Rather, the information contained in the
sequence(s) is used as the starting material to create a "second
generation" sequence(s) derived from the original sequence(s) and
then the "second generation" sequence(s) is prepared and expressed
as a protein.
[0239] Accordingly, in another embodiment, the invention provides a
method for preparing an anti-IFNAR-1 antibody comprising:
[0240] (a) providing: (i) a heavy chain variable region antibody
sequence comprising a CDR1 sequence selected from the group
consisting of SEQ ID NOs: 1, 2, 3 and 4, a CDR2 sequence selected
from the group consisting of SEQ ID NOs: 5, 6, 7, and 8 and/or a
CDR3 sequence selected from the group consisting of SEQ ID NOs: 9,
10, 11, and 12; and (ii) a light chain variable region antibody
sequence comprising a CDR1 sequence selected from the group
consisting of SEQ ID NOs: 13, 14, 15, and 16, a CDR2 sequence
selected from the group consisting of SEQ ID NOs: 17, 18, 19, and
20 and/or a CDR3 sequence selected from the group consisting of SEQ
ID NOs: 21, 22, 23, and 24;
[0241] (b) altering at least one amino acid residue within the
first antibody sequence and/or the second antibody sequence to
create at least one altered antibody sequence; and
[0242] (c) preparing the altered antibody sequence; and
[0243] (d) expressing the altered antibody sequence as a
protein.
[0244] Standard molecular biology techniques can be used to prepare
and express the altered antibody sequence.
[0245] Preferably, the antibody encoded by the altered antibody
sequence(s) is one that retains one, some or all of the functional
properties of the anti-IFNAR-1 antibodies described herein, which
functional properties include, but are not limited to:
[0246] (i) binding to IFNAR-1;
[0247] (ii) inhibiting the binding of type I interferons to
IFNAR-1;
[0248] (iii) binding to live cells expressing human IFNAR-1;
[0249] (iv) binding to human IFNAR-1 with a K.sub.D of 10.sup.-8 M
or less (e.g., 10.sup.-9 M or 10.sup.-10 M or less);
[0250] (v) binding to a unique epitope on IFNAR-1 (to eliminate the
possibility that monoclonal antibodies with complimentary
activities when used in combination would compete for binding to
the same epitope).
[0251] The functional properties of the altered antibodies can be
assessed using standard assays available in the art and/or
described herein. For example, the ability of the antibody to bind
IFNAR-1 can be determined using standard binding assays, such as
those set forth in the Examples (e.g., ELISAs).
[0252] In certain embodiments of the methods of engineering
antibodies of the invention, mutations can be introduced randomly
or selectively along all or part of an anti-IFNAR-1 antibody coding
sequence (e.g., 3F11, 4G5, 11E2, or 9D4 coding sequence) and the
resulting modified anti-IFNAR-1 antibodies can be screened for
binding activity and/or other functional properties as described
herein. Mutational methods have been described in the art. For
example, PCT Publication WO 02/092780 by Short describes methods
for creating and screening antibody mutations using saturation
mutagenesis, synthetic ligation assembly, or a combination thereof.
Alternatively, PCT Publication WO 03/074679 by Lazar et al.
describes methods of using computational screening methods to
optimize physiochemical properties of antibodies.
Nucleic Acid Molecules Encoding Antibodies of the Invention
[0253] Another aspect of the invention pertains to nucleic acid
molecules that encode the antibodies of the invention. The nucleic
acids may be present in whole cells, in a cell lysate, or in a
partially purified or substantially pure form. A nucleic acid is
"isolated" or "rendered substantially pure" when purified away from
other cellular components or other contaminants, e.g., other
cellular nucleic acids or proteins, by standard techniques,
including alkaline/SDS treatment, CsCl banding, column
chromatography, agarose gel electrophoresis and others well known
in the art. See, F. Ausubel, et al., ed. (1987) Current Protocols
in Molecular Biology, Greene Publishing and Wiley Interscience, New
York. A nucleic acid of the invention can be, for example, DNA or
RNA and may or may not contain intronic sequences. In a preferred
embodiment, the nucleic acid is a cDNA molecule.
[0254] Nucleic acids of the invention can be obtained using
standard molecular biology techniques. For antibodies expressed by
hybridomas (e.g., hybridomas prepared from transgenic mice carrying
human immunoglobulin genes as described further below), cDNAs
encoding the light and heavy chains of the antibody made by the
hybridoma can be obtained by standard PCR amplification or cDNA
cloning techniques. For antibodies obtained from an immunoglobulin
gene library (e.g., using phage display techniques), nucleic acid
encoding the antibody can be recovered from the library.
[0255] Preferred nucleic acids molecules of the invention are those
encoding the VH and VL sequences of the 3F11, 4G5, 11E2, and 9D4
monoclonal antibodies. DNA sequences encoding the 3F11 VH and VL
sequences are shown in SEQ ID NOs: 33 and 37, respectively. DNA
sequences encoding the 4G5 VH and VL sequences are shown in SEQ ID
NOs: 34 and 38, respectively. DNA sequences encoding the 11E2 VH
and VL sequences are shown in SEQ ID NOs: 35 and 39, respectively.
DNA sequences encoding the 9D4 VH and VL sequences are shown in SEQ
ID NOs: 36 and 40, respectively.
[0256] Once DNA fragments encoding VH and VL segments are obtained,
these DNA fragments can be further manipulated by standard
recombinant DNA techniques, for example to convert the variable
region genes to full-length antibody chain genes, to Fab fragment
genes or to a scFv gene. In these manipulations, a VL- or
VH-encoding DNA fragment is operatively linked to another DNA
fragment encoding another protein, such as an antibody constant
region or a flexible linker. The term "operatively linked", as used
in this context, is intended to mean that the two DNA fragments are
joined such that the amino acid sequences encoded by the two DNA
fragments remain in-frame.
[0257] The isolated DNA encoding the VH region can be converted to
a full-length heavy chain gene by operatively linking the
VH-encoding DNA to another DNA molecule encoding heavy chain
constant regions (CH1, CH2 and CH3). The sequences of human heavy
chain constant region genes are known in the art (see e.g., Kabat,
E. A., et al. (1991) Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The heavy chain constant region can be an IgG1,
IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most
preferably is an IgG1 or IgG4 constant region. For a Fab fragment
heavy chain gene, the VH-encoding DNA can be operatively linked to
another DNA molecule encoding only the heavy chain CH1 constant
region.
[0258] The isolated DNA encoding the VL region can be converted to
a full-length light chain gene (as well as a Fab light chain gene)
by operatively linking the VL-encoding DNA to another DNA molecule
encoding the light chain constant region, CL. The sequences of
human light chain constant region genes are known in the art (see
e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The light chain constant region can be a kappa or
lambda constant region, but most preferably is a kappa constant
region.
[0259] To create a scFv gene, the VH- and VL-encoding DNA fragments
are operatively linked to another fragment encoding a flexible
linker, e.g., encoding the amino acid sequence
(Gly.sub.4-Ser).sub.3, such that the VH and VL sequences can be
expressed as a contiguous single-chain protein, with the VL and VH
regions joined by the flexible linker (see e.g., Bird et al. (1988)
Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci.
USA 85:5879-5883; McCafferty et al., (1990) Nature
348:552-554).
Production of Monoclonal Antibodies of the Invention
[0260] Monoclonal antibodies (mAbs) of the present invention can be
produced by a variety of techniques, including conventional
monoclonal antibody methodology e.g., the standard somatic cell
hybridization technique of Kohler and Milstein (1975) Nature 256:
495. Although somatic cell hybridization procedures are preferred,
in principle, other techniques for producing monoclonal antibody
can be employed e.g., viral or oncogenic transformation of B
lymphocytes.
[0261] The preferred animal system for preparing hybridomas is the
murine system. Hybridoma production in the mouse is a very
well-established procedure. Immunization protocols and techniques
for isolation of immunized splenocytes for fusion are known in the
art. Fusion partners (e.g., murine myeloma cells) and fusion
procedures are also known.
[0262] Chimeric or humanized antibodies of the present invention
can be prepared based on the sequence of a murine monoclonal
antibody prepared as described above. DNA encoding the heavy and
light chain immunoglobulins can he obtained from the murine
hybridoma of interest and engineered to contain non-murine (e.g.,
human) immunoglobulin sequences using standard molecular biology
techniques. For example, to create a chimeric antibody, the murine
variable regions can be linked to human constant regions using
methods known in the art (see e.g., U.S. Pat. No. 4,816,567 to
Cabilly et al.). To create a humanized antibody, the murine CDR
regions can be inserted into a human framework using methods known
in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S.
Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et
al.).
[0263] In a preferred embodiment, the antibodies of the invention
are human monoclonal antibodies. Such human monoclonal antibodies
directed against IFNAR-1 can be generated using transgenic or
transchromosomic mice carrying parts of the human immune system
rather than the mouse system. These transgenic and transchromosomic
mice include mice referred to herein as HuMAb mice and KM mice,
respectively, and are collectively referred to herein as "human Ig
mice."
[0264] The HuMAb mouse.RTM. (Medarex, Inc.) contains human
immunoglobulin gene miniloci that encode unrearranged human heavy
(.mu. and .gamma.) and .kappa. light chain immunoglobulin
sequences, together with targeted mutations that inactivate the
endogenous .mu. and .kappa. chain loci (see e.g., Lonberg, et al.
(1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit
reduced expression of mouse IgM or .kappa., and in response to
immunization, the introduced human heavy and light chain transgenes
undergo class switching and somatic mutation to generate high
affinity human IgG.kappa. monoclonal (Lonberg, N. et al. (1994),
supra; reviewed in Lonberg, N. (1994) Handbook of Experimental
Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern.
Rev. Immunol. 13: 65-93, and Harding, F. and Lonberg, N. (1995)
Ann. NY. Acad. Sci. 764:536-546). The preparation and use of HuMab
mice, and the genomic modifications carried by such mice, is
further described in Taylor, L. et al. (1992) Nucleic Acids
Research 20:6287-6295; Chen, J. et al. (1993) International
Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad.
Sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics
4:117-123; Chen, J. et at (1993) EMBO J. 12: 821-830; Tuaillon et
al. (1994) J. Immunol. 152:2912-2920; Taylor, L. et al. (1994)
International Immunology 6: 579-591; and Fishwild, D. et al. (1996)
Nature Biotechnology 14: 845-851, the contents of all of which are
hereby specifically incorporated by reference in their entirety.
See further, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126;
5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299;
and 5,770,429; all to Lonberg and Kay; U.S. Pat. No. 5,545,807 to
Surani et al.; PCT Publication Nos. WO 92/03918, WO 93/12227, WO
94/25585, WO 97/13852, WO 98/24884 and WO 99/45962, all to Lonberg
and Kay; and PCT Publication No. WO 01/14424 to Korman et al.
[0265] In another embodiment, human antibodies of the invention can
be raised using a mouse that carries human immunoglobulin sequences
on transgenes and transchomosomes, such as a mouse that carries a
human heavy chain transgene and a human light chain
transchromosome. Such mice, referred to herein as "KM mice", are
described in detail in PCT Publication WO 02/43478 to Ishida et
al.
[0266] Still further, alternative transgenic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise anti-IFNAR-1 antibodies of the invention. For
example, an alternative transgenic system referred to as the
Xenomouse (Abgenix, Inc.) can be used; such mice are described in,
for example, U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6,
150,584 and 6,162,963 to Kucherlapati et al.
[0267] Moreover, alternative transchromosomic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise anti-IFNAR-1 antibodies of the invention. For
example, mice carrying both a human heavy chain transchromosome and
a human light chain tranchromosome, referred to as "TC mice" can be
used; such mice are described in Tomizuka et al. (2000) Proc. Natl.
Acad. Sci. USA 97:722-727. Furthermore, cows carrying human heavy
and light chain transchromosomes have been described in the art
(Kuroiwa et al. (2002) Nature Biotechnology 20:889-894) and can be
used to raise anti-IFNAR-1 antibodies of the invention.
[0268] Human monoclonal antibodies of the invention can also be
prepared using phage display methods for screening libraries of
human immunoglobulin genes. Such phage display methods for
isolating human antibodies are established in the art. See for
example: U.S. Pat. Nos. 5,223,409; 5,403,484; and 5,571,698 to
Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et
al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty et al.;
and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313;
6,582,915 and 6,593,081 to Griffiths et al.
[0269] Human monoclonal antibodies of the invention can also be
prepared using SCID mice into which human immune cells have been
reconstituted such that a human antibody response can be generated
upon immunization. Such mice are described in, for example, U.S.
Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.
Immunization of Human Ig Mice
[0270] When human Ig mice are used to raise human antibodies of the
invention, such mice can be immunized with a purified or enriched
preparation of IFNAR-1 antigen and/or cells expressing IFNAR-1, as
described by Lonberg, N. et al. (1994) Nature 368(6474): 856-859;
Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851; and
PCT Publication WO 98/24884 and WO 01/14424. Preferably, the mice
will be 6-16 weeks of age upon the first infusion. For example, a
purified or enriched preparation (5-50 .mu.g) of IFNAR-1 antigen
can be used to immunize the human Ig mice intraperitoneally. In the
event that immunizations using a purified or enriched preparation
of IFNAR-1 antigen do not result in antibodies, mice can also be
immunized with cells expressing IFNAR-1, e.g., a human T-cell line,
to promote immune responses.
[0271] Detailed procedures to generate fully human monoclonal
antibodies to IFNAR-1 are described in Example 1 below. Cumulative
experience with various antigens has shown that the transgenic mice
respond when initially immunized intraperitoneally (IP) with
antigen in complete Freund's adjuvant, followed by every other week
IP immunizations (up to a total of 6) with antigen in incomplete
Freund's adjuvant. However, adjuvants other than Freund's are also
found to be effective. In addition, whole cells in the absence of
adjuvant are found to be highly immunogenic. The immune response
can be monitored over the course of the immunization protocol with
plasma samples being obtained by retroorbital bleeds. The plasma
can be screened by ELISA (as described below), and mice with
sufficient titers of anti-IFNAR-1 human immunoglobulin can be used
for fusions. Mice can be boosted intravenously with antigen 3 days
before sacrifice and removal of the spleen. It is expected that 2-3
fusions for each immunization may need to be performed. Between 6
and 24 mice are typically immunized for each antigen. Usually both
HCo7 and HCo12 strains are used. In addition, both HCo7 and HCo12
transgene can be bred together into a single mouse having two
different human heavy chain transgenes (HCo7/HCo12).
Generation of Hybridomas Producing Human Monoclonal Antibodies of
the Invention
[0272] To generate hybridomas producing human monoclonal antibodies
of the invention, splenocytes and/or lymph node cells from
immunized mice can be isolated and fused to an appropriate
immortalized cell line, such as a mouse myeloma cell line. The
resulting hybridomas can be screened for the production of
antigen-specific antibodies. For example, single cell suspensions
of splenic lymphocytes from immunized mice can be fused to
one-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma
cells (ATCC, CRL 1580) with 50% PEG. Cells are plated at
approximately 2.times.10.sup.5 in flat bottom microtiter plate,
followed by a two week incubation in selective medium containing
20% fetal Clone Serum, 18% "653" conditioned media, 5% origen
(IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5mM HEPES, 0.055 mM
2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin,
50 mg/ml gentamycin and 1.times. HAT (Sigma; the HAT is added 24
hours after the fusion). After approximately two weeks, cells can
be cultured in medium in which the HAT is replaced with HT.
Individual wells can then be screened by ELISA for human monoclonal
IgM and IgG antibodies. Once extensive hybridoma growth occurs,
medium can be observed usually after 10-14 days. The antibody
secreting hybridomas can be replated, screened again, and if still
positive for human IgG, the monoclonal antibodies can be subcloned
at least twice by limiting dilution. The stable subclones can then
be cultured in vitro to generate small amounts of antibody in
tissue culture medium for characterization.
[0273] To purify human monoclonal antibodies, selected hybridomas
can be grown in two-liter spinner-flasks for monoclonal antibody
purification. Supernatants can be filtered and concentrated before
affinity chromatography with protein A-sepharose (Pharmacia,
Piscataway, N.J.). Eluted IgG can be checked by gel electrophoresis
and high performance liquid chromatography to ensure purity. The
buffer solution can be exchanged into PBS, and the concentration
can be determined by OD280 using 1.43 extinction coefficient. The
monoclonal antibodies can be aliquoted and stored at -80.degree.
C.
Generation of Transfectomas Producing Monoclonal Antibodies of the
Invention
[0274] Antibodies of the invention also can be produced in a host
cell transfectoma using, for example, a combination of recombinant
DNA techniques and gene transfection methods as is well known in
the art (e.g., Morrison, S. (1985) Science 229:1202).
[0275] For example, to express the antibodies, or antibody
fragments thereof; DNAs encoding partial or full-length light and
heavy chains, can be obtained by standard molecular biology
techniques (e.g., PCR amplification or cDNA cloning using a
hybridoma that expresses the antibody of interest) and the DNAs can
be inserted into expression vectors such that the genes are
operatively linked to transcriptional and translational control
sequences. In this context, the term "operatively linked" is
intended to mean that an antibody gene is ligated into a vector
such that transcriptional and translational control sequences
within the vector serve their intended function of regulating the
transcription and translation of the antibody gene. The expression
vector and expression control sequences are chosen to be compatible
with the expression host cell used. The antibody light chain gene
and the antibody heavy chain gene can be inserted into separate
vector or, more typically, both genes are inserted into the same
expression vector. The antibody genes are inserted into the
expression vector by standard methods (e.g., ligation of
complementary restriction sites on the antibody gene fragment and
vector, or blunt end ligation if no restriction sites are present).
The light and heavy chain variable regions of the antibodies
described herein can be used to create full-length antibody genes
of any antibody isotype by inserting them into expression vectors
already encoding heavy chain constant and light chain constant
regions of the desired isotype such that the V.sub.H segment is
operatively linked to the C.sub.H segment(s) within the vector and
the V.sub.L segment is operatively linked to the C.sub.L segment
within the vector. Additionally or alternatively, the recombinant
expression vector can encode a signal peptide that facilitates
secretion of the antibody chain from a host cell. The antibody
chain gene can be cloned into the vector such that the signal
peptide is linked in-frame to the amino terminus of the antibody
chain gene. The signal peptide can be an immunoglobulin signal
peptide or a heterologous signal peptide (i.e., a signal peptide
from a non-immunoglobulin protein).
[0276] In addition to the antibody chain genes, the recombinant
expression vectors of the invention carry regulatory sequences that
control the expression of the antibody chain genes in a host cell.
The term "regulatory sequence" is intended to include promoters,
enhancers and other expression control elements (e.g.,
polyadenylation signals) that control the transcription or
translation of the antibody chain genes. Such regulatory sequences
are described, for example, in Goeddel (Gene Expression Technology.
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990)). It will be appreciated by those skilled in the art that
the design of the expression vector, including the selection of
regulatory sequences, may depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. Preferred regulatory sequences for mammalian host
cell expression include viral elements that direct high levels of
protein expression in mammalian cells, such as promoters and/or
enhancers derived from cytomegalovirus (CMV), Simian Virus 40
(SV40), adenovirus, (e.g., the adenovirus major late promoter
(AdMLP) and polyoma. Alternatively, nonviral regulatory sequences
may be used, such as the ubiquitin promoter or .beta.-globin
promoter. Still further, regulatory elements composed of sequences
from different sources, such as the SR.alpha. promoter system,
which contains sequences from the SV40 early promoter and the long
terminal repeat of human T cell leukemia virus type 1 (Takebe, Y.
et al. (1988) Mol. Cell. Biol. 8:466-472).
[0277] In addition to the antibody chain genes and regulatory
sequences, the recombinant expression vectors of the invention may
carry additional sequences, such as sequences that regulate
replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017, all by Axel et al.). For example, typically the
selectable marker gene confers resistance to drugs, such as G418,
hygromycin or methotrexate, on a host cell into which the vector
has been introduced. Preferred selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells
with methotrexate selection/amplification) and the neo gene (for
G418 selection).
[0278] For expression of the light and heavy chains, the expression
vector(s) encoding the heavy and light chains is transfected into a
host cell by standard techniques. The various forms of the term
"transfection" are intended to encompass a wide variety of
techniques commonly used for the introduction of exogenous DNA into
a prokaryotic or eukaryotic host cell, e.g., electroporation,
calcium-phosphate precipitation, DEAE-dextran transfection and the
like. Although it is theoretically possible to express the
antibodies of the invention in either prokaryotic or eukaryotic
host cells, expression of antibodies in eukaryotic cells, and most
preferably mammalian host cells, is the most preferred because such
eukaryotic cells, and in particular mammalian cells, are more
likely than prokaryotic cells to assemble and secrete a properly
folded and immunologically active antibody. Prokaryotic expression
of antibody genes has been reported to be ineffective for
production of high yields of active antibody (Boss, M. A. and Wood,
C. R. (1985) Immunology Today 6:12-13).
[0279] Preferred mammalian host cells for expressing the
recombinant antibodies of the invention include Chinese Hamster
Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub
and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used
with a DHFR selectable marker, e.g., as described in R. J. Kaufman
and P. A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells,
COS cells and SP2 cells. In particular, for use with NSO myeloma
cells, another preferred expression system is the GS gene
expression system disclosed in WO 87/04462, WO 89/01036 and EP
338,841. When recombinant expression vectors encoding antibody
genes are introduced into mammalian host cells, the antibodies are
produced by culturing the host cells for a period of time
sufficient to allow for expression of the antibody in the host
cells or, more preferably, secretion of the antibody into the
culture medium in which the host cells are grown. Antibodies can be
recovered from the culture medium using standard protein
purification methods.
Characterization of Antibody Binding to Antigen
[0280] Antibodies of the invention can be tested for binding to
IFNAR-1 by, for example, standard ELISA. Briefly, microtiter plates
are coated with purified IFNAR-1 at 0.25 .mu.g/ml in PBS, and then
blocked with 5% bovine serum albumin in PBS. Dilutions of antibody
(e.g., dilutions of plasma from IFNAR-1-immunized mice) are added
to each well and incubated for 1-2 hours at 37.degree. C. The
plates are washed with PBS/Tween and then incubated with secondary
reagent (e.g., for human antibodies, a goat-anti-human IgG
Fc-specific polyclonal reagent) conjugated to alkaline phosphatase
for 1 hour at 37.degree. C. After washing, the plates are developed
with pNPP substrate (1 mg/ml), and analyzed at OD of 405-650.
Preferably, mice which develop the highest titers will be used for
fusions.
[0281] An ELISA assay as described above can also be used to screen
for hybridomas that show positive reactivity with IFNAR-1
immunogen. Hybridomas that bind with high avidity to IFNAR-1 are
subcloned and further characterized. One clone from each hybridoma,
which retains the reactivity of the parent cells (by ELISA), can be
chosen for making a 5-10 vial cell bank stored at -140 .degree. C.,
and for antibody purification.
[0282] To purify anti-IFNAR-1 antibodies, selected hybridomas can
be grown in two-liter spinner-flasks for monoclonal antibody
purification. Supernatants can be filtered and concentrated before
affinity chromatography with protein A-sepharose (Pharmacia,
Piscataway, N.J.). Eluted IgG can be checked by gel electrophoresis
and high performance liquid chromatography to ensure purity. The
buffer solution can be exchanged into PBS, and the concentration
can be determined by OD.sub.280 using 1.43 extinction coefficient.
The monoclonal antibodies can be aliquoted and stored at
-80.degree. C.
[0283] To determine if the selected anti-IFNAR-1 monoclonal
antibodies bind to unique epitopes, each antibody can be
biotinylated using commercially available reagents (Pierce,
Rockford, Ill.). Competition studies using unlabeled monoclonal
antibodies and biotinylated monoclonal antibodies can be performed
using IFNAR-1 coated-ELISA plates as described above. Biotinylated
mAb binding can be detected with a strep-avidin-alkaline
phosphatase probe.
[0284] To determine the isotype of purified antibodies, isotype
ELISAs can be performed using reagents specific for antibodies of a
particular isotype. For example, to determine the isotype of a
human monoclonal antibody, wells of microtiter plates can be coated
with 1 .mu.g/ml of anti-human immunoglobulin overnight at 4.degree.
C. After blocking with 1% BSA, the plates are reacted with 1 .mu.g
/ml or less of test monoclonal antibodies or purified isotype
controls, at ambient temperature for one to two hours. The wells
can then be reacted with either human IgG1 or human IgM-specific
alkaline phosphatase-conjugated probes. Plates are developed and
analyzed as described above.
[0285] To demonstrate binding of monoclonal antibodies to live
cells expressing IFNAR-1, flow cytometry can be used. Briefly, cell
lines expressing IFNAR-1 (grown under standard growth conditions)
are mixed with various concentrations of monoclonal antibodies in
PBS containing 0.1% BSA and 10% fetal calf serum, and incubated at
37.degree. C. for 1 hour. After washing, the cells are reacted with
Fluorescein-labeled anti-human IgG antibody under the same
conditions as the primary antibody staining. The samples can be
analyzed by FACScan instrument using light and side scatter
properties to gate on single cells. An alternative assay using
fluorescence microscopy may be used (in addition to or instead of)
the flow cytometry assay. Cells can be stained exactly as described
above and examined by fluorescence microscopy. This method allows
visualization of individual cells, but may have diminished
sensitivity depending on the density of the antigen.
[0286] Anti-IFNAR-1 human IgGs can be further tested for reactivity
with IFNAR-1 antigen by Western blotting. Briefly, cell extracts
from cells expressing IFNAR-1 can be prepared and subjected to
sodium dodecyl sulfate polyacrylamide gel electrophoresis. After
electrophoresis, the separated antigens are transferred to
nitrocellulose membranes, blocked with 10% fetal calf serum, and
probed with the monoclonal antibodies to be tested. Human IgG
binding can be detected using anti-human IgG alkaline phosphatase
and developed with BCIP/NBT substrate tablets (Sigma Chem. Co., St.
Louis, Mo.).
Immunoconjugates
[0287] In another aspect, the present invention features an
anti-IFNAR-1 antibody, or a fragment thereof, conjugated to a
therapeutic moiety, such as a cytotoxin, a drug (e.g., an
immunosuppressant) or a radiotoxin. Such conjugates are referred to
herein as "immunoconjugates". Immunoconjugates that include one or
more cytotoxins are referred to as "immunotoxins." A cytotoxin or
cytotoxic agent includes any agent that is detrimental to (e.g.,
kills) cells. Examples include taxol, cytochalasin B, gramicidin D,
ethidium bromide, emetine, mitomycin, etoposide, tenoposide,
vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,
dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin
D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. Therapeutic agents also include, for example,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine).
[0288] Other preferred examples of therapeutic cytotoxins that can
be conjugated to an antibody of the invention include duocarmycins,
calicheamicins, maytansines and auristatins, and derivatives
thereof. An example of a calicheamicin antibody conjugate is
commercially available (Mylotarg.TM.; Wyeth-Ayerst).
[0289] Cytoxins can be conjugated to antibodies of the invention
using linker technology available in the art. Examples of linker
types that have been used to conjugate a cytotoxin to an antibody
include, but are not limited to, hydrazones, thioethers, esters,
disulfides and peptide-containing linkers. A linker can be chosen
that is, for example, susceptible to cleavage by low pH within the
lysosomal compartment or susceptible to cleavage by proteases, such
as proteases preferentially expressed in tumor tissue such as
cathepsins (e.g., cathepsins B, C, D).
[0290] For further discussion of types of cytotoxins, linkers and
methods for conjugating therapeutic agents to antibodies, see also
Saito, G. et al. (2003) Adv. Drug Deliv. Rev. 55:199-215; Trail, P.
A. et al. (2003) Cancer Immunol. Immunother. 52:328-337; Payne, G.
(2003) Cancer Cell 3:207-212; Allen, T. M. (2002) Nat. Rev. Cancer
2:750-763; Pastan, I. and Kreitman, R. J. (2002) Curr. Opin.
Investig. Drugs 3:1089-1091; Senter, P. D. and Springer, C. J.
(2001) Adv. Drug Deliv. Rev. 53:247-264.
[0291] Antibodies of the present invention also can be conjugated
to a radioactive isotope to generate cytotoxic
radiopharmaceuticals, also referred to as radioimmunoconjugates.
Examples of radioactive isotopes that can be conjugated to
antibodies for use diagnostically or therapeutically include, but
are not limited to, iodine.sup.131, indium.sup.111, yttrium.sup.90
and lutetium.sup.177. Method for preparing radioimmunconjugates are
established in the art. Examples of radioimmunoconjugates are
commercially available, including Zevalin.TM. (IDEC
Pharmaceuticals) and Bexxar.TM. (Corixa Pharmaceuticals), and
similar methods can be used to prepare radioimmunoconjugates using
the antibodies of the invention.
[0292] The antibody conjugates of the invention can be used to
modify a given biological response, and the drug moiety is not to
be construed as limited to classical chemical therapeutic agents.
For example, the drug moiety may be a protein or polypeptide
possessing a desired biological activity. Such proteins may
include, for example, an enzymatically active toxin, or active
fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor or
interferon-.gamma.; or, biological response modifiers such as, for
example, lymphokines, interleukin-1 ("IL-1"), interleukin-2
("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony
stimulating factor ("GM-CSF"), granulocyte colony stimulating
factor ("G-CSF"), or other growth factors.
[0293] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Amon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies `84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982).
Bispecific Molecules
[0294] In another aspect, the present invention features bispecific
molecules comprising an anti-IFNAR-1 antibody, or a fragment
thereof, of the invention. An antibody of the invention, or
antigen-binding portions thereof, can be derivatized or linked to
another functional molecule, e.g., another peptide or protein
(e.g., another antibody or ligand for a receptor) to generate a
bispecific molecule that binds to at least two different binding
sites or target molecules. The antibody of the invention may in
fact be derivatized or linkd to more than one other functional
molecule to generate multispecific molecules that bind to more than
two different binding sites and/or target molecules; such
multispecific molecules are also intended to be encompassed by the
term "bispecific molecule" as used herein. To create a bispecific
molecule of the invention, an antibody of the invention can be
functionally linked (e.g., by chemical coupling, genetic fusion,
noncovalent association or otherwise) to one or more other binding
molecules, such as another antibody, antibody fragment, peptide or
binding mimetic, such that a bispecific molecule results.
[0295] Accordingly, the present invention includes bispecific
molecules comprising at least one first binding specificity for
IFNAR-1 and a second binding specificity for a second target
epitope. In a particular embodiment of the invention, the second
target epitope is an Fc receptor, e.g., human Fc.gamma.RI (CD64) or
a human Fc.alpha. receptor (CD89). Therefore, the invention
includes bispecific molecules capable of binding both to
Fc.gamma.R, Fc.alpha.R or Fc.epsilon.R expressing effector cells
(e.g., monocytes, macrophages or polymorphonuclear cells (PMNs)),
and to target cells expressing IFNAR-1. These bispecific molecules
target IFNAR-1 expressing cells to effector cell and trigger Fc
receptor-mediated effector cell activities, such as phagocytosis of
an IFNAR-1 expressing cells, antibody dependent cell-mediated
cytotoxicity (ADCC), cytokine release, or generation of superoxide
anion.
[0296] In an embodiment of the invention in which the bispecific
molecule is multispecific, the molecule can further include a third
binding specificity, in addition to an anti-Fc binding specificity
and an anti-IFNAR-1 binding specificity. In one embodiment, the
third binding specificity is an anti-enhancement factor (EF)
portion, e.g., a molecule which binds to a surface protein involved
in cytotoxic activity and thereby increases the immune response
against the target cell. The "anti-enhancement factor portion" can
be an antibody, functional antibody fragment or a ligand that binds
to a given molecule, e.g., an antigen or a receptor, and thereby
results in an enhancement of the effect of the binding determinants
for the F.sub.c receptor or target cell antigen. The
"anti-enhancement factor portion" can bind an F.sub.c receptor or a
target cell antigen. Alternatively, the anti-enhancement factor
portion can bind to an entity that is different from the entity to
which the first and second binding specificities bind. For example,
the anti-enhancement factor portion can bind a cytotoxic T-cell
(e.g. via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune
cell that results in an increased immune response against the
target cell).
[0297] In one embodiment, the bispecific molecules of the invention
comprise as a binding specificity at least one antibody, or an
antibody fragment thereof, including, e.g., an Fab, Fab',
F(ab').sub.2, Fv, or a single chain Fv. The antibody may also be a
light chain or heavy chain dimer, or any minimal fragment thereof
such as a Fv or a single chain construct as described in Ladner et
al. U.S. Pat. No. 4,946,778, the contents of which is expressly
incorporated by reference.
[0298] In one embodiment, the binding specificity for an Fc.gamma.
receptor is provided by a monoclonal antibody, the binding of which
is not blocked by human immunoglobulin G (IgG). As used herein, the
term "IgG receptor" refers to any of the eight .gamma.-chain genes
located on chromosome 1. These genes encode a total of twelve
transmembrane or soluble receptor isoforms which are grouped into
three Fc.gamma. receptor classes: Fc.gamma.RI (CD64),
Fc.gamma.RII(CD32), and Fc.gamma.RIII (CD16). In one preferred
embodiment, the Fc.gamma. receptor a human high affinity
Fc.gamma.RI. The human Fc.gamma.RI is a 72 kDa molecule, which
shows high affinity for monomeric IgG (10.sup.8-10.sup.9
M.sup.-1).
[0299] The production and characterization of certain preferred
anti-Fc.gamma. monoclonal antibodies are described by Fanger et al.
in PCT Publication WO 88/00052 and in U.S. Pat. No. 4,954,617, the
teachings of which are fully incorporated by reference herein.
These antibodies bind to an epitope of Fc.gamma.RI, Fc.gamma.RII or
Fc.gamma.RIII at a site which is distinct from the Fc.gamma.
binding site of the receptor and, thus, their binding is not
blocked substantially by physiological levels of IgG. Specific
anti-Fc.gamma.RI antibodies useful in this invention are mAb 22,
mAb 32, mAb 44, mAb 62 and mAb 197. The hybridoma producing mAb 32
is available from the American Type Culture Collection, ATCC
Accession No. HB9469. In other embodiments, the anti-Fc.gamma.
receptor antibody is a humanized form of monoclonal antibody 22
(H22). The production and characterization of the H22 antibody is
described in Graziano, R. F. et al. (1995) J. Immunol 155 (10):
4996-5002 and PCT Publication WO 94/10332. The H22 antibody
producing cell line was deposited at the American Type Culture
Collection under the designation HA022CL1 and has the accession no.
CRL 11177.
[0300] In still other preferred embodiments, the binding
specificity for an Fc receptor is provided by an antibody that
binds to a human IgA receptor, e.g., an Fc-alpha receptor
(Fc.alpha.RI (CD89)), the binding of which is preferably not
blocked by human immunoglobulin A (IgA). The term "IgA receptor" is
intended to include the gene product of one a-gene (FcaRI) located
on chromosome 19. This gene is known to encode several
alternatively spliced transmembrane isoforms of 55 to 110 kDa.
Fc.alpha.RI (CD89) is constitutively expressed on
monocytes/macrophages, eosinophilic and neutrophilic granulocytes,
but not on non-effector cell populations. Fc.alpha.RI has medium
affinity (.apprxeq.5.times.10.sup.7 M.sup.-1) for both IgA1 and
IgA2, which is increased upon exposure to cytokines such as G-CSF
or GM-CSF (Morton, H. C. et al. (1996) Critical Reviews in
Immunology 16:423-440). Four Fc.alpha.RI-specific monoclonal
antibodies, identified as A3, A59, A62 and A77, which bind
Fc.alpha.RI outside the IgA ligand binding domain, have been
described (Monteiro, R. C. et al. (1992) J. Immunol. 148:1764).
[0301] Fc.alpha.RI and Fc.gamma.RI are preferred trigger receptors
for use in the bispecific molecules of the invention because they
are (1) expressed primarily on immune effector cells, e.g.,
monocytes, PMNs, macrophages and dendritic cells; (2) expressed at
high levels (e.g., 5,000-100,000 per cell); (3) mediators of
cytotoxic activities (e.g., ADCC, phagocytosis); (4) mediate
enhanced antigen presentation of antigens, including self-antigens,
targeted to them.
[0302] While human monoclonal antibodies are preferred, other
antibodies which can be employed in the bispecific molecules of the
invention are murine, chimeric and humanized monoclonal
antibodies.
[0303] The bispecific molecules of the present invention can be
prepared by conjugating the constituent binding specificities,
e.g., the anti-FcR and anti-IFNAR-1 binding specificities, using
methods known in the art. For example, each binding specificity of
the bispecific molecule can be generated separately and then
conjugated to one another. When the binding specificities are
proteins or peptides, a variety of coupling or cross-linking agents
can be used for covalent conjugation. Examples of cross-linking
agents include protein A, carbodiimide,
N-succinimidyl-S-acetyl-thioacetate (SATA),
5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide
(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and
sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate
(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med.
160:1686; Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA
82:8648). Other methods include those described in Paulus (1985)
Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science
229:81-83), and Glennie et al. (1987) J. Immunol. 139: 2367-2375).
Preferred conjugating agents are SATA and sulfo-SMCC, both
available from Pierce Chemical Co. (Rockford, Ill.).
[0304] When the binding specificities are antibodies, they can be
conjugated via sulfhydryl bonding of the C-terminus hinge regions
of the two heavy chains. In a particularly preferred embodiment,
the hinge region is modified to contain an odd number of sulfhydryl
residues, preferably one, prior to conjugation.
[0305] Alternatively, both binding specificities can be encoded in
the same vector and expressed and assembled in the same host cell.
This method is particularly useful where the bispecific molecule is
a mAb x mAb, mAb x Fab, Fab x F(ab').sub.2 or ligand x Fab fusion
protein. A bispecific molecule of the invention can be a single
chain molecule comprising one single chain antibody and a binding
determinant, or a single chain bispecific molecule comprising two
binding determinants. Bispecific molecules may comprise at least
two single chain molecules. Methods for preparing bispecific
molecules are described for example in U.S. Pat. No. 5,260,203;
U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat. No.
5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S.
Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No.
5,482,858.
[0306] Binding of the bispecific molecules to their specific
targets can be confirmed by, for example, enzyme-linked
immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis,
bioassay (e.g., growth inhibition), or Western Blot assay. Each of
these assays generally detects the presence of protein-antibody
complexes of particular interest by employing a labeled reagent
(e.g., an antibody) specific for the complex of interest. For
example, the FcR-antibody complexes can be detected using e.g., an
enzyme-linked antibody or antibody fragment which recognizes and
specifically binds to the antibody-FcR complexes. Alternatively,
the complexes can be detected using any of a variety of other
immunoassays. For example, the antibody can be radioactively
labeled and used in a radioimmunoassay (RIA) (see, for exam*,
Weintraub, B., Principles of Radioimmunoassays, Seventh Training
Course on Radioligand Assay Techniques, The Endocrine Society,
March, 1986, which is incorporated by reference herein). The
radioactive isotope can be detected by such means as the use of a y
counter or a scintillation counter or by autoradiography.
Pharmaceutical Compositions
[0307] In another aspect, the present invention provides a
composition, e.g., a pharmaceutical composition, containing one or
a combination of monoclonal antibodies, or antigen-binding
portion(s) thereof, of the present invention, formulated together
with a pharmaceutically acceptable carrier. Such compositions may
include one or a combination of (e.g., two or more different)
antibodies, or immunoconjugates or bispecific molecules of the
invention. For example, a pharmaceutical composition of the
invention can comprise a combination of antibodies (or
immunoconjugates or bispecifics) that bind to different epitopes on
the target antigen or that have complementary activities.
[0308] Pharmaceutical compositions of the invention also can be
administered in combination therapy, i.e., combined with other
agents. For example, the combination therapy can include an
anti-IFNAR-1 antibody of the present invention combined with at
least one other immunosuppressing agent.
[0309] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
Preferably, the carrier is suitable for intravenous, intramuscular,
subcutaneous, parenteral, spinal or epidermal administration (e.g.,
by injection or infusion). Depending on the route of
administration, the active compound, i.e., antibody,
immunoconjuage, or bispecific molecule, may be coated in a material
to protect the compound from the action of acids and other natural
conditions that may inactivate the compound.
[0310] The pharmaceutical compounds of the invention may include
one or more pharmaceutically acceptable salts. A "pharmaceutically
acceptable salt" refers to a salt that retains the desired
biological activity of the parent compound and does not impart any
undesired toxicological effects (see e.g., Berge, S. M., et al.
(1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid
addition salts and base addition salts. Acid addition salts include
those derived from nontoxic inorganic acids, such as hydrochloric,
nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous
and the like, as well as from nontoxic organic acids such as
aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic
acids, hydroxy alkanoic acids, aromatic acids, aliphatic and
aromatic sulfonic acids and the like. Base addition salts include
those derived from alkaline earth metals, such as sodium,
potassium, magnesium, calcium and the like, as well as from
nontoxic organic amines, such as N,N'-dibenzylethylenediamine,
N-methylglucamine, chloroprocaine, choline, diethanolamine,
ethylenediamine, procaine and the like.
[0311] A pharmaceutical composition of the invention also may
include a pharmaceutically acceptable anti-oxidant. Examples of
pharmaceutically acceptable antioxidants include: (1) water soluble
antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium
bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)
oil-soluble antioxidants, such as ascorbyl palmitate, butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin,
propyl gallate, alpha-tocopherol, and the like; and (3) metal
chelating agents, such as citric acid, ethylenediamine tetraacetic
acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the
like.
[0312] Examples of suitable aqueous and nonaqueous carriers that
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0313] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures, supra, and by the inclusion of
various antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monostearate and gelatin.
[0314] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. The use
of such media and agents for pharmaceutically active substances is
known in the art. Except insofar as any conventional media or agent
is incompatible with the active compound, use thereof in the
pharmaceutical compositions of the invention is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0315] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent that
delays absorption, for example, monostearate salts and gelatin.
[0316] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying (lyophilization) that yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0317] The amount of active ingredient which can be combined with a
carrier material to produce a single dosage form will vary
depending upon the subject being treated, and the particular mode
of administration. The amount of active ingredient which can be
combined with a carrier material to produce a single dosage form
will generally be that amount of the composition which produces a
therapeutic effect. Generally, out of one hundred per cent, this
amount will range from about 0.01 per cent to about ninety-nine
percent of active ingredient, preferably from about 0.1 per cent to
about 70 per cent, most preferably from about 1 per cent to about
30 per cent of active ingredient in combination with a
pharmaceutically acceptable carrier.
[0318] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0319] For administration of the antibody, the dosage ranges from
about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the
host body weight. For example dosages can be 0.3 mg/kg body weight,
1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10
mg/kg body weight or within the range of 1-10 mg/kg. An exemplary
treatment regime entails administration once per week, once every
two weeks, once every three weeks, once every four weeks, once a
month, once every 3 months or once every three to 6 months.
Preferred dosage regimens for an anti-IFNAR-1 antibody of the
invention include 1 mg/kg body weight or 3 mg/kg body weight via
intravenous administration, with the antibody being given using one
of the following dosing schedules: (i) every four weeks for six
dosages, then every three months; (ii) every three weeks; (iii) 3
mg/kg body weight once followed by 1 mg/kg body weight every three
weeks.
[0320] In some methods, two or more monoclonal antibodies with
different binding specificities are administered simultaneously, in
which case the dosage of each antibody administered falls within
the ranges indicated. Antibody is usually administered on multiple
occasions. Intervals between single dosages can be, for example,
weekly, monthly, every three months or yearly. Intervals can also
be irregular as indicated by measuring blood levels of antibody to
the target antigen in the patient. In some methods, dosage is
adjusted to achieve a plasma antibody concentration of about 1-1000
.mu.g/ml and in some methods about 25-300 .mu.g/ml.
[0321] Alternatively, antibody can be administered as a sustained
release formulation, in which case less frequent administration is
required. Dosage and frequency vary depending on the half-life of
the antibody in the patient. In general, human antibodies show the
longest half life, followed by humanized antibodies, chimeric
antibodies, and nonhuman antibodies. The dosage and frequency of
administration can vary depending on whether the treatment is
prophylactic or therapeutic. In prophylactic applications, a
relatively low dosage is administered at relatively infrequent
intervals over a long period of time. Some patients continue to
receive treatment for the rest of their lives. In therapeutic
applications, a relatively high dosage at relatively short
intervals is sometimes required until progression of the disease is
reduced or terminated, and preferably until the patient shows
partial or complete amelioration of symptoms of disease.
Thereafter, the patient can be administered a prophylactic
regime.
[0322] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, or the ester, salt or amide thereof, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular compositions employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts.
[0323] A "therapeutically effective dosage" of an anti-IFNAR-1
antibody of the invention preferably results in a decrease in
severity of disease symptoms, an increase in frequency and duration
of disease symptom-free periods, or a prevention of impairment or
disability due to the disease affliction. In the case of, for
example, Systemic Lupus Erythematosus (SLE), a therapeutically
effective dose preferably prevents further deterioration of
physical symptoms associated with SLE, such as, for example, pain,
fatigue or weakness. A therapeutically effective dose preferably
also prevents or delays onset of SLE, such as may be desired when
early or preliminary signs of the disease are present. Likewise it
includes delaying chronic progression associated with SLE.
Laboratory tests utilized in the diagnosis of SLE include
chemistries, hematology, serology and radiology. Accordingly, any
clinical or biochemical assay that monitors any of the foregoing
may be used to determine whether a particular treatment is a
therapeutically effective dose for treating SLE. One of ordinary
skill in the art would be able to determine such amounts based on
such factors as the subject's size, the severity of the subject's
symptoms, and the particular composition or route of administration
selected.
[0324] A composition of the present invention can be administered
via one or more routes of administration using one or more of a
variety of methods known in the art. As will be appreciated by the
skilled artisan, the route and/or mode of administration will vary
depending upon the desired results. Preferred routes of
administration for antibodies of the invention include intravenous,
intramuscular, intradermal, intraperitoneal, subcutaneous, spinal
or other parenteral routes of administration, for example by
injection or infusion. The phrase "parenteral administration" as
used herein means modes of administration other than enteral and
topical administration, usually by injection, and includes, without
limitation, intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal, epidural
and infrasternal injection and infusion.
[0325] Alternatively, an antibody of the invention can be
administered via a non-parenteral route, such as a topical,
epidermal or mucosal route of administration, for example,
intranasally, orally, vaginally, rectally, sublingually or
topically.
[0326] The active compounds can be prepared with carriers that will
protect the compound against rapid release, such as a controlled
release formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in
the art. See, e.g., Sustained and Controlled Release Drug Delivery
Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,
1978.
[0327] Therapeutic compositions can be administered with medical
devices known in the art. For example, in a preferred embodiment, a
therapeutic composition of the invention can be administered with a
needleless hypodermic injection device, such as the devices
disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335;
5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of
well-known implants and modules useful in the present invention
include: U.S. Pat. No. 4,487,603, which discloses an implantable
micro-infusion pump for dispensing medication at a controlled rate;
U.S. Pat. No. 4,486,194, which discloses a therapeutic device for
administering medicants through the skin; U.S. Pat. No. 4,447,233,
which discloses a medication infusion pump for delivering
medication at a precise infusion rate; U.S. Pat. No. 4,447,224,
which discloses a variable flow implantable infusion apparatus for
continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses
an osmotic drug delivery system having multi-chamber compartments;
and U.S. Pat. No. 4,475,196, which discloses an osmotic drug
delivery system. These patents are incorporated herein by
reference. Many other such implants, delivery systems, and modules
are known to those skilled in the art.
[0328] In certain embodiments, the human monoclonal antibodies of
the invention can be formulated to ensure proper distribution in
vivo. For example, the blood-brain barrier (BBB) excludes many
highly hydrophilic compounds. To ensure that the therapeutic
compounds of the invention cross the BBB (if desired), they can be
formulated, for example, in liposomes. For methods of manufacturing
liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and
5,399,331. The liposomes may comprise one or more moieties which
are selectively transported into specific cells or organs, thus
enhance targeted drug delivery (see, e.g., V. V. Ranade (1989) J.
Clin. Pharmacol. 29:685). Exemplary targeting moieties include
folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et
al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res.
Commun. 153:1038); antibodies (P. G. Bloeman et at (1995) FEBS
Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother.
39:180); surfactant protein A receptor (Briscoe et al. (1995) Am.
J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem.
269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett.
346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods
4:273.
Uses and Methods of the Invention
[0329] The antibodies (and immunoconjugates and bispecific
molecules) of the present invention have in vitro and in vivo
diagnostic and therapeutic utilities. For example, these molecules
can be administered to cells in culture, e.g. in vitro or ex vivo,
or in a subject, e.g., in vivo, to treat, prevent or diagnose a
variety of disorders. The term "subject" as used herein in intended
to includes human and non-human animals. Non-human animals includes
all vertebrates, e.g., mammals and non-mammals, such as non-human
primates, sheep, dogs, cats, cows, horses, chickens, amphibians,
and reptiles. The methods are particularly suitable for treating
human patients having a disorder associated with aberrant or
inappropriate Type I interferon expression (e.g.,
overexpression).
[0330] When antibodies to IFNAR-1 are administered together with
another agent, the two can be administered in either order or
simultaneously. For example, an anti-IFNAR-1 antibody of the
invention can be used in combination with one or more of the
following agents: anti-IFN.alpha. antibody, anti-IFN.gamma.
receptor antibody, soluble IFN.gamma. receptor, anti-TNF antibody,
anti-TNF receptor antibody and/or soluble TNF receptor (see e.g.,
U.S. Pat. No. 5,888,511). Furthermore, an anti-IFNAR-1 antibody of
invention can be used in combination with a Flt3 ligand antagonist
(see e.g., U.S. Application No. 2002/0160974).
[0331] In one embodiment, the antibodies (and immunoconjugates and
bispecific molecules) of the invention can be used to detect levels
of IFNAR-1, or levels of cells that express IFNAR-1. This can be
achieved, for example, by contacting a sample (such as an in vitro
sample) and a control sample with the anti-IFNAR-1 antibody under
conditions that allow for the formation of a complex between the
antibody and IFNAR-1. Any complexes formed between the antibody and
IFNAR-1 are detected and compared in the sample and the control.
For example, standard detection methods, well-known in the art,
such as ELISA and flow cytometic assays, can be performed using the
compositions of the invention.
[0332] Accordingly, in one aspect, the invention further provides
methods for detecting the presence of IFNAR-1 (e.g., human IFNAR-1
antigen) in a sample, or measuring the amount of IFNAR-1,
comprising contacting the sample, and a control sample, with an
antibody of the invention, or an antigen binding portion thereof,
which specifically binds to IFNAR-1, under conditions that allow
for formation of a complex between the antibody or portion thereof
and IFNAR-1. The formation of a complex is then detected, wherein a
difference in complex formation between the sample compared to the
control sample is indicative of the presence of IFNAR-1 in the
sample.
[0333] Also within the scope of the invention are kits comprising
the compositions (e.g., antibodies, human antibodies,
immunoconjugates and bispecific molecules) of the invention and
instructions for use. The kit can further contain a least one
additional reagent, or one or more additional antibodies of the
invention (e.g., an antibody having a complementary activity which
binds to an epitope on the target antigen distinct from the first
antibody). Kits typically include a label indicating the intended
use of the contents of the kit. The term label includes any
writing, or recorded material supplied on or with the kit, or which
otherwise accompanies the kit.
[0334] IFNAR-1 is part of the cellular receptor for Type I
interferons, and Type I interferons are known to be
immunoregulatory cytokines that are involved in, inter cilia, T
cell differentiation, antibody production and activity and survival
of memory T cells. Moreover, increased expression of Type I
interferons has been described in numerous autoimmune diseases, in
HIV infection, in transplant rejection and in graft versus host
disease (GVHD). Accordingly, the anti-IFNAR-1 antibodies (and
immunoconjugates and bispecific molecules) of the invention, which
inhibit the functional activity of Type I interferons, can be used
in a variety of clinical indications involving aberrant or
undesired Type I interferon activity. The invention, therefore,
provides a method of inhibiting a Type I interferon-mediated
disease or disorder, wherein the method comprises administering an
antibody, or antigen-binding portion thereof, of the invention (or
immunconjugate or bispecific molecule of the invention) such that
the Type I interferon-mediated disease or disorder is treated.
[0335] Specific examples of autoimmune conditions in which the
antibodies of the invention can be used include, but are not
limited to, the following: systemic lupus erythematosus (SLE),
insulin dependent diabetes mellitus (IDDM), inflammatory bowel
disease (IBD) (including Crohn's Disease, Ulcerative Colitis and
Celiac's Disease), multiple sclerosis (MS), psoriasis, autoimmune
thyroiditis, rheumatoid arthritis (RA) and glomerulonephritis.
Furthermore, the antibody compositions of the invention can be used
for inhibiting or preventing transplant rejection or in the
treatment of graft versus host disease (GVHD) or in the treatment
of HIV infection/AIDS.
[0336] High levels of IFN.alpha. have been observed in the serum of
patients with systemic lupus erythematosus (SLE) (see e.g., Kim et
al. (1987) Clin. Exp. Immunol. 70:562-569). Moreover,
administration of IFN.alpha., for example in the treatment of
cancer or viral infections, has been shown to induce SLE
(Garcia-Porrua et al. (1998) Clin. Exp. Rheumatol. 16:107-108).
Accordingly, in another embodiment, the anti-IFNAR-1 antibodies of
the invention can be used in the treatment of SLE by administering
the antibody to a subject in need of treatment. The antibody can be
used alone or in combination with other anti-SLE agents, such as
non-steroidal anti-inflammatory drugs (NSAIDs), analgesics,
corticosteroids (e.g., predinisone, hydrocortisone),
immunosuppressants (such as cyclophosphamide, azathioprine, and
methotrexate), antimalarials (such as hydroxychloroquine) and
biologic drugs that inhibit the production of dsDNA antibodies
(e.g., LJP 394).
[0337] IFN.alpha. also has been implicated in the pathology of Type
I diabetes. For example, the presence of immunoreactive IFN.alpha.
in pancreatic beta cells of Type I diabetes patients has been
reported (Foulis et al. (1987) Lancet 2:1423-1427). Prolonged use
of IFN.alpha. in anti-viral therapy also has been shown to induce
Type I diabetes (Waguri et al. (1994) Diabetes Res. Clin. Pract.
23:33-36). Accordingly, in another embodiment, the anti-IFNAR-1
antibodies of the invention can be used in the treatment of Type I
diabetes by administering the antibody to a subject in need of
treatment. The antibody can be used alone or in combination with
other anti-diabetic agents, such as insulin.
[0338] Antibodies to IFNAR have been shown to be effective in an
animal model of inflammatory bowel disease (see US Patent
Application 60/465,155). Thus, the anti-IFNAR-1 antibodies of the
invention can be used in the treatment of inflammatory bowel
disease (IBD), including ulcerative colitis and Crohn's disease, by
administering the antibody to a subject in need of treatment. The
antibody can be used alone or in combination with other anti-IBD
agents, such as drugs containing mesalamine (including
sulfasalazine and other agents containing 5-aminosalicylic acid
(5-ASA), such as olsalazine and balsalazide), non-steroidal
anti-inflammatory drugs (NSAIDs), analgesics, corticosteroids
(e.g., predinisone, hydrocortisone), TNF-inhibitors (including
adilimumab (Humira.RTM.), etanercept (Enbrel.RTM.) and infliximab
(Remicade.RTM.)), immunosuppressants (such as 6-mercaptopurine,
azathioprine and cyclosporine A), and antibiotics.
[0339] Treatment with IFN.alpha. has also been observed to induce
autoimmune thyroiditis (Monzani et al. (2004) Clin. Exp. Med.
3:199-210; Prummel and Laurberg (2003) Thyroid 13:547-551).
Accordingly, in another embodiment, the anti-IFNAR antibodies of
the invention can be used in the treatment of autoimmune thyroid
disease, including autoimmune primary hypothyroidism, Graves'
Disease, Hashimoto's thyroiditis and destructive thyroiditis with
hypothyroidism, by administering the antibody to a subject in need
of treatment. The antibody can be used alone or in combination with
other agents or treatments, such as anti-thyroid drugs, radioactive
iodine and subtotal thyroidectomy.
[0340] Increased levels of type I interferons, especially
IFB-.beta., have been observed in the serum of patients with RA
(see e.g., Hertzog et al. (1988) Clin. Immunol. Immunopath.
48:192). Thus, in an embodiment, the anti-IFNAR-1 antibodies of the
present invention can be used in the treatment of RA by
administering the antibody to a subject in need of such treatment.
The antibody can be used alone or in combination with one or more
other anti-RA agent, such as a non-steroidal anti-inflammatory drug
(NSAID), a COX-2 inhibitor, an analgesic, a corticosteroid (e.g.,
predinisone, hydrocortisone), gold, an immunosuppressant (e.g.,
methotrexate), a B-cell depletion agent (e.g., Rituxan.TM.), a
B-cell agonist (e.g., LymphoStat-B.TM.) and an anti-TNF-.alpha.
agent (e.g., EMBREL.TM., HUMIRA.RTM. and REMICADE.TM.).
[0341] Administration of IFN.alpha. has been reported to exacerbate
psoriasis. Accordingly, in another embodiment, the anti-IFNAR-1
antibodies of the invention can be used in the treatment of
psoriasis and psoriatic arthritis by administering the antibody to
a subject in need of such treatment. The antibody can be used alone
or in combination with one or more other anti-psoriasis treatments
such as phototherapy, topical therapy (e.g., topical
glucocorticoids), or systemic therapy (e.g., methotrexate, a
synthetic retinoid, cyclosporine), an anti-TNF-.alpha. agent (e.g.,
EMBREL.TM., HUMIRA.RTM. and REMICADE.TM.), and a T-cell inhibitor
(e.g., Raptiva.TM.).
[0342] High levels of IFNa also have been observed in the
circulation of patients with HIV infection and its presence is a
predictive marker of AIDS progression (DeStefano et al. (1982) J.
Infec. Disease 146:451; Vadhan-Raj et al. (1986) Cancer Res.
46:417). Thus, in another embodiment, an anti-IFNAR-1 antibody of
the invention is used in the treatment of HIV infection or AIDS by
administering the antibody to a subject in need of treatment. The
antibody can be used alone or in combination with other anti-HIV
agents, such as nucleoside reverse transcriptase inhibitors,
non-nucleoside reverse transcriptase inhibitors, protease
inhibitors and fusion inhibitors.
[0343] Antibodies to IFNAR-1 have been demonstrated to be effective
in inhibiting allograft rejection and prolonging allograft survival
(see e.g., Tovey et al. (1996) J. Leukoc. Biol. 59:512-517; Benizri
et al. (1998) J. Interferon Cytokine Res. 18:273-284). Accordingly,
the anti-IFNAR-1 antibodies of the invention also can be used in
transplant recipients to inhibit allograft rejection and/or prolong
allograft survival. The invention provides a method of inhibiting
transplant rejection by administering an anti-IFNAR-1 antibody of
the invention to a transplant recipient in need of treatment.
Examples of tissue transplants that can be treated include, but are
not limited to, liver, lung, kidney, heart, small bowel, and
pancreatic islet cells, as well as the treatment of graft versus
host disease (GVHD). The antibody can be used alone or in
combination with other agents for inhibiting transplant rejection,
such as immunosuppressive agents (e.g., cyclosporine, azathioprine,
methylprednisolone, prednisolone, prednisone, mycophenolate
mofetil, sirilimus, rapamycin, tacrolimus), anti-infective agents
(e.g., acyclovir, clotrimazole, ganciclovir, nystatin,
trimethoprimsulfarnethoxazole), diuretics (e.g., bumetanide,
furosemide, metolazone) and ulcer medications (e.g., cimetidine,
famotidine, lansoprazole, omeprazole, ranitidine, sucralfate).
[0344] The present invention is further illustrated by the
following examples which should not be construed as further
limiting. The contents of all figures and all references, patents
and published patent applications cited throughout this application
are expressly incorporated herein by reference.
Example 1
Generation of Human Monoclonal Antibodies Against IFNAR-1
Antigen
[0345] Soluble IFNAR-1, containing the extracellular domain of
IFNAR-1 was generated by recombinant methods and used as antigen
for immunization.
Transgenic HuMab Mice
[0346] Fully human monoclonal antibodies to IFNAR-1 were prepared
using HCo7, HCo12, and HCo7.times.HCo12 strains of HuMab transgenic
mice, each of which express human antibody genes. In each of these
mouse strains, the endogenous mouse kappa light chain gene has been
homozygously disrupted as described in Chen et al. (1993) EMBO J.
12:811-820 and the endogenous mouse heavy chain gene has been
homozygously disrupted as described in Example 1 of PCT Publication
WO 01/09187. Each of these mouse strains carries a human kappa
light chain transgene, KCo5, as described in Fishwild at at (1996)
Nature Biotechnology 14:845-851. The HCo7 strain carries the HCo7
human heavy chain transgene as described in U.S. Pat. Nos.
5,545,806; 5,625,825; and 5,545,807. The HCo12 strain carries the
HCo12 human heavy chain transgene as described in Example 2 of PCT
Publication WO 01/09187. The HCo7.times.HCo 12 stain carries both
the HCo7 and the HCo 12 transgenes and was made by breeding the two
strains together.
HuMab Mice Immunizations:
[0347] To generate fully human monoclonal antibodies to IFNAR-1,
HuMab mice were immunized with purified recombinant IFNAR-1 as
antigen. General immunization schemes for HuMab mice are described
in Lonberg, N. et al (1994) Nature 368(6474): 856-859; Fishwild, D.
at al. (1996) Nature Biotechnology 14: 845-851 and PCT Publication
WO 98/24884. The mice were 6-16 weeks of age upon the first
infusion of antigen. A purified recombinant preparation (5-50
.mu.g) of soluble IFNAR-1 antigen was used to immunize the HuMab
mice intraperitonealy, subcutaneously (Sc) or via footpad
injection.
[0348] Transgenic mice were immunized twice with antigen in
complete Freund's adjuvatnt or Ribi adjuvant either
intraperitonealy (IP), subcutaneously (Sc) or via footpad (FP),
followed by 3-21 days IP, Sc or FP immunization (up to a total of
11 immunizations) with the antigen in incomplete Freund's or Ribi
adjuvant. The immune response was monitored by retroorbital bleeds.
The plasma was screened by ELISA (as described below), and mice
with sufficient titers of anti-IFNAR-1 human immunogolobulin were
used for fusions. Mice were boosted intravenously with antigen 3
and 2 days before sacrifice and removal of the spleen. Typically,
10-35 fusions for each antigen were performed. Several dozen mice
were immunized for each antigen.
Selection of HuMab Mice Producing Anti-IFNAR-1 Antibodies:
[0349] To select HuMab mice producing antibodies that bound
IFNAR-1, sera from immunized mice was tested by ELISA as described
by Fishwild, D. et al. (1996). Briefly, microtiter plates were
coated with purified recombinant IFNAR-1 from E. coli at 1-2
.mu.g/ml in PBS, 50 .mu.l/wells incubated 4.degree. C. overnight
then blocked with 200 .mu.l/well of 5% chicken serum in PBS/Tween
(0.05%). Dilutions of plasma from IFNAR-1-immunized mice were added
to each well and incubated for 1-2 hours at ambient temperature.
The plates were washed with PBS/Tween and then incubated with a
goat-anti-human IgG Fc polyclonal antibody conjugated with
horseradish peroxidase (HRP) for 1 hour at room temperature. After
washing, the plates were developed with ABTS substrate (Sigma,
A-1888, 0.22 mg/ml) and analyzed by spectrophotometer at OD
415-495. Mice that developed the highest titers of anti-IFNAR-1
antibodies were used for fusions. Fusions were performed as
described below and hybridoma supernatants were tested for
anti-IFNAR-1 activity by ELISA.
Generation of Hybridomas Producing Human Monoclonal Antibodies to
IFNAR-1:
[0350] The mouse splenocytes, isolated from the HuMab mice, were
fused with PEG to a mouse myeloma cell line based upon standard
protocols. The resulting hybridomas were then screened for the
production of antigen-specific antibodies. Single cell suspensions
of splenic lymphocytes from immunized mice were fused to one-fourth
the number of SP2/0 nonsecreting mouse myeloma cells (ATCC, CRL
1581) with 50% PEG (Sigma). Cells were plated at approximately
1.times.10.sup.5/well in flat bottom microtiter plate, followed by
about two week incubation in selective medium containing 10% fetal
bovine serum, 10% P388D1 (ATCC, CRL TIB-63) conditioned medium,
3-5% origen (IGEN) in DMEM (Mediatech, CRL 10013, with high
glucose, L-glutamine and sodium pyruvate) plus 5 mM HEPES, 0.055 mM
2-mercaptoethanol, 50 mg/ml gentamycin and 1.times. HAT (Sigma, CRL
P-7185). After 1-2 weeks, cells were cultured in medium in which
the HAT was replaced with HT. Individual Wells were then screened
by ELISA (described above) for human anti-IFNAR-1 monoclonal IgG
antibodies. Once extensive hybridoma growth occurred, medium was
monitored usually after 10-14 days. The antibody secreting
hybridomas were replated, screened again and, if still positive for
human IgG, anti-IFNAR-1 monoclonal antibodies were subcloned at
least twice by limiting dilution. The stable subclones were then
cultured in vitro to generate small amounts of antibody in tissue
culture medium for further characterization.
[0351] Hybridoma clones 3F11, 4G5, 11E2, and 9D4 were selected for
further analysis.
Example 2
Structural Characterization of Human Monoclonal Antibodies 3F11,
4G5, 11E2, and 9D4
[0352] The cDNA sequences encoding the heavy and light chain
variable regions of the 3F11, 4G5, 11E2, and 9D4 monoclonal
antibodies were obtained from the 3F11, 4G5, 11 E2, and 9D4
hybridomas, respectively, using standard PCR techniques and were
sequenced using standard DNA sequencing techniques.
[0353] The nucleotide and amino acid sequences of the heavy chain
variable region of 3F11 are shown in FIG. 1A and in SEQ ID NO: 33
and 25, respectively.
[0354] The nucleotide and amino acid sequences of the light chain
variable region of 3F11 are shown in FIG. 1B and in SEQ ID NO: 37
and 29, respectively.
[0355] Comparison of the 3F11 heavy chain immunoglobulin sequence
to the known human germline immunoglobulin heavy chain sequences
demonstrated that the 3F11 heavy chain utilizes a VH segment from
human germline VH 4-34, an undetermined D segment, and a JH segment
from human germline JH 6b. The alignment of the 3F11 VH sequence to
the germline VH 4-34 sequence is shown in FIG. 5. Further analysis
of the 3F11 VH sequence using the Kabat system of CDR region
determination led to the delineation of the heavy chain CDR1, CDR2
and CD3 regions as shown in FIGS. 1A and 5, and in SEQ ID NOs: 1, 5
and 9, respectively.
[0356] Comparison of the 3F11 light chain immunoglobulin sequence
to the known human germline immunoglobulin light chain sequences
demonstrated that the 3F11 light chain utilizes a VL segment from
human germline VK L18 and a JK segment from human germline JK 5.
The alignment of the 3F11 VL sequence to the germline VK L18
sequence is shown in FIG. 8. Further analysis of the 3F11 VL
sequence using the Kabat system of CDR region determination led to
the delineation of the light chain CDR1, CDR2 and CD3 regions as
shown in FIGS. 1B and 8, and in SEQ ID NOs:13, 17 and 21,
respectively.
[0357] The nucleotide and amino acid sequences of the heavy chain
variable region of 4G5 are shown in FIG. 2A and in SEQ ID NO: 34
and 26, respectively.
[0358] The nucleotide and amino acid sequences of the light chain
variable region of 4G5 are shown in FIG. 2B and in SEQ ID NO: 38
and 30, respectively.
[0359] Comparison of the 4G5 heavy chain immunoglobulin sequence to
the known human germline immunoglobulin heavy chain sequences
demonstrated that the 4G5 heavy chain utilizes a VH segment from
human germline VH 4-34, an undetermined D segment, and a JH segment
from human germline JH 4b. The alignment of the 4G5 VH sequence to
the germline VH 4-34 sequence is shown in FIG. 6. Further analysis
of the 4G5 VH sequence using the Kabat system of CDR region
determination led to the delineation of the heavy chain CDR1, CDR2
and CD3 regions as shown in FIGS. 2A and 6, and in SEQ ID NOs: 2, 6
and 10, respectively.
[0360] Comparison of the 4G5 light chain immunoglobulin sequence to
the known human germline immunoglobulin light chain sequences
demonstrated that the 4G5 light chain utilizes a VL segment from
human germline VK L18 and a JK segment from human germline JK 2.
The alignment of the 4G5 VL sequence to the germline VK L18
sequence is shown in FIG. 9. Further analysis of the 4G5 VL
sequence using the Kabat system of CDR region determination led to
the delineation of the light chain CDR1, CDR2 and CD3 regions as
shown in FIGS. 2B and 9, and in SEQ ID NOs:14, 18 and 22,
respectively.
[0361] The nucleotide and amino acid sequences of the heavy chain
variable region of 11E2 are shown in FIG. 3A and in SEQ ID NO: 35
and 27, respectively.
[0362] The nucleotide and amino acid sequences of the light chain
variable region of 11E2 are shown in FIG. 3B and in SEQ ID NO: 39
and 31, respectively.
[0363] Comparison of the 11E2 heavy chain immunoglobulin sequence
to the known human germline immunoglobulin heavy chain sequences
demonstrated that the 11E2 heavy chain was derived from, or is
highly similar to, a VH segment from human germline VH 5-51, an
undetermined D segment, and a JH segment from human germline JH 4b.
The alignment of the 11E2 VH sequence to the germline VH 5-51
sequence is shown in FIG. 7. Further analysis of the 11E2 VH
sequence using the Kabat system of CDR region determination led to
the delineation of the heavy chain CDR1, CDR2 and CD3 regions as
shown in FIGS. 3A and 7, and in SEQ ID NOs: 3, 7 and 11,
respectively.
[0364] Comparison of the 11E2 light chain immunoglobulin sequence
to the known human germline immunoglobulin light chain sequences
demonstrated that the 11E2 light chain utilizes a VL segment from
human germline VK A27 and a JK segment from human germline JK 5.
The alignment of the 11E2 VL sequence to the germline VK A27
sequence is shown in FIG. 10. Further analysis of the 11E2 VL
sequence using the Kabat system of CDR region determination led to
the delineation of the light chain CDR1, CDR2 and CD3 regions as
shown in FIGS. 3B and 10, and in SEQ ID NOs:15, 19 and 23,
respectively.
[0365] The nucleotide and amino acid sequences of the heavy chain
variable region of 9D4 are shown in FIG. 4A and in SEQ ID NO: 36
and 28, respectively.
[0366] The nucleotide and amino acid sequences of the light chain
variable region of 9D4 are shown in FIG. 4B and in SEQ ID NO: 40
and 32, respectively.
[0367] Comparison of the 9D4 heavy chain immunoglobulin sequence to
the known human germline immunoglobulin heavy chain sequences
demonstrated that the 9D4 heavy chain was derived from, or is
highly similar to, a VH segment from human germline VH 5-51, an
undetermined D segment, and a JH segment from human germline JH 4b.
The alignment of the 9D4 VH sequence to the germline VH 5-51
sequence is shown in FIG. 7. Further analysis of the 9D4 VH
sequence using the Kabat system of CDR region determination led to
the delineation of the heavy chain CDR1, CDR2 and CD3 regions as
shown in FIGS. 4A and 7, and in SEQ ID NOs: 4, 8 and 12,
respectively.
[0368] Comparison of the 9D4 light chain immunoglobulin sequence to
the known human germline immunoglobulin light chain sequences
demonstrated that the 9D4 light chain utilizes a VL segment from
human germline VK A27 and a JK segment from human germline JK 5.
The alignment of the 9D4 VL sequence to the germline VK A27
sequence is shown in FIG. 10. Further analysis of the 9D4 VL
sequence using the Kabat system of CDR region determination led to
the delineation of the light chain CDR1, CDR2 and CD3 regions as
shown in FIGS. 3B and 10, and in SEQ ID NOs:16, 20 and 24,
respectively.
Example 3
Anti-IFNAR-1 Human Monoclonal Antibodies Inhibit the Biological
Activity of Interferon .alpha.2b
[0369] The cell line Daudi, derived from a human B-lymphoblast
Burkitt's lymphoma, expresses high levels of IFNAR-1, and the
growth of these cells is inhibited by Type I interferons. To
measure the functional blocking ability of human anti-IFNAR-1
antibodies, two different assays were performed, a cell
proliferation assay and a reporter assay.
[0370] In the first assay, Daudi cells were cultured with
interferon .alpha.2b in the presence or absence of antibody and
proliferation was measured by uptake of .sup.3[H]-thymidine. Daudi
cells (ATCC CCL-213) were grown in RPMI containing 10% FCS, and 2
mM beta mercaptoethanol (media). Cells were spun and resuspended at
a concentration of 1.times.10.sup.6 cells/ml in media with added 1%
human serum albumin (media & HS). To each well of a 96-well
plate, 100 .mu.l of 200 U/ml interferon .alpha.2b (Schering
Corporation) containing the appropriate concentration of antibody
was added. 100 .mu.l of Daudi cells in media & HS were added to
the wells and the plates were incubated for 48 hours at 37.degree.
C. The plates were pulsed with 1 .mu.Ci of .sup.3[H]-thymidine and
incubated for an additional 24 hours. The plates were harvested,
collected onto a 96-well fiber filter plate, and counted using a
TopCount scintillation counter (Packard). The counts per minute
were plotted as a function of antibody concentration and the data
was analyzed by non-linear regression, sigmoidal dose-response
(variable slope) using the Prism software (San Diego, Calif.).
[0371] In the second assay, U937 cells were transfected with a
construct in which an Interferon Stimulated Response Element was
linked to a reporter gene (ISRE-RG) and the ability of humanized
anti-IFNAR-1 antibodies to block IFN-induced expression of the
reporter gene was measured. The cells were grown in RPMI containing
10% FCS, and 2 mM beta mercaptoethanol (media). The cells
(1.times.10.sup.6 cells/ml) were resuspended in media with added 2%
human serum. 100 .mu.l of cells was added to a 96-well plate.
Antibodies were serially diluted in media containing 200 U/ml of
interferon .alpha.2b (Schering corporation) and 100 .mu.l was added
to each well. The plates were incubated overnight at 37.degree. C.
Following this incubation, expression of the reporter gene was
assessed by flow cytometry. Geometric mean fluorescent intensity
was plotted as a function of antibody concentration and the data
was analyzed by non-linear regression, sigmoidal dose-response
(variable slope) using the Prism software (San Diego, Calif.).
[0372] Using the above described two assays, the potency of the
3F11 human monoclonal antibody was compared to the murine
anti-IFNAR-1 antibody 64G12 (ECACC Deposit No. 92022605) and to the
humanized anti-IFNAR-1 antibody D1 H3K1 (described further in U.S.
Ser. No. 60/465,058). The potency of 3F11 showed a 5-10 fold
greater potency than the mouse antibody and a 6-30 fold greater
potency than the humanized antibody. The results are summarized in
Table 1 below.
TABLE-US-00001 TABLE 1 Blocking ability of human anti-IFNAR-1
antibody on IFN alpha 2b Cell Proliferation ISRE-RG Reporter
Isotype (Daudi) EC.sub.50 (nM) (U937) EC.sub.50 (nM) 64G12 m IgG1
3.1 6.0 DI H3K1 h IgG1 9.3 8.0 3F11 h IgG1 0.3 1.2
Example 4
Anti-IFNAR-1 Human Monoclonal Antibodies Inhibit the Biological
Activity of IFN Omega
[0373] Using the Daudi proliferation assay described above in
Example 3, the ability of the human anti-1FNAR-1 antibody to
inhibit IFN omega responses was tested. To each well of a 96-well
plate, 100 .mu.l of 200 U/ml interferon omega (PBL) containing the
appropriate concentration of antibody was added. The human
antibodies 3F11, 4G5, 11E2, and 9D4 were 4-18 times more potent (as
measured by EC.sub.50) than the mouse 64G12 antibody. The results
are summarized in Table 2 below.
TABLE-US-00002 TABLE 2 Blocking ability of human anti-IFNAR-1
antibody on IFN omega Cell Proliferation Isotype (Daudi) EC.sub.50
(nM) 64G12 m IgG1 5.5 DI H3K1 h IgG1 30.7 3F11 h IgG1 0.6 4G5 h
IgG1 1.4 11E2 h IgG1 0.3 9D4 h IgG1 0.3
Example 5
Anti-IFNAR-1 Human Monoclonal Antibodies Inhibit the Biological
Activity of Multiple Type I IFNs
[0374] As described in Example 3, interferon alpha inhibits the
proliferation of Daudi (Burkitts lymphoma, ATCC #CCL-213) cells in
a dose dependant manner. A neutralizing antibody, which blocks
interferon binding to its receptor, will restore proliferation.
Using this cell proliferation assay, the specificity of the
purified human anti-IFN alpha antibodies was examined by testing
for blockade of natural lymphoblastoid IFN.alpha., natural
leukocyte interferon, 13 recombinant IFN alpha subtypes, IFN beta
and IFN omega.
[0375] Daudi cells were grown in culture medium (RPMI 1640
supplemented with 10% FCS, 1.times.2-ME, L-glutamine and penicillin
streptomycin) with and without the addition of IFNa in a 96 well,
flat-bottomed cell culture plate. Each type I interferon tested was
assayed at EC.sub.50 and mixed with a 2-fold serial titration of
anti-IFNAR-1 antibody 3F11, typically from 50 ug/ml (312 nM)
through 381 pg/ml (2.4 pM). The antibody/IFN mixture was added to
Daudi cells in a 96-well bottomed plate to a final density of
1.times.10.sup.4 Daudi cells/100 ul/well and incubated at
37.degree. C., 5% CO.sub.2, 72 hrs. Proliferation was assayed with
the addition of MTS (Promega), 20 ul/well, and O.D. at 490 nm was
measured following a further 3 hour incubation. The viable cell
number was proportional to the O.D. reading. Percentage blockade of
interferon was calculated relative to Daudi proliferation in the
absence of IFN (=100% blockade) and in the presence of IFN alone
(=0% blockade). The 3F11 antibody was scored according to the
degree of blockade, resulting in a profile of IFNa subtype
specificity. The results demonstrated that the human
anti-interferon alpha receptor 1 antibody 3F11 inhibits the action
of multiple interferon alpha subtypes, including IFN.alpha. 6, 2b,
2a, 1, 16, 10, 8, 5, 14, 17, 7, 4, and 21, as well as
lymphoblastoid IFN, leukocyte IFN, and IFN omega. 3F11 is a lower
level inhibitor of IFN beta, although inhibition of greater than
50% was observed. The % blockade and standard deviation of
interferon are shown in Table 3, below.
TABLE-US-00003 TABLE 3 Antibody Inhibition of Multiple type I
interferons 3F11 IFN Blockade (%) at 1000x Ab IFN mean sd
Lymphoblastoid IFN 94.9 2.9 IFN.alpha. 6 107.1 6.6 IFN.alpha. 2b
101.9 0.4 IFN.alpha. 2a 103.1 3.0 IFN.alpha. 1 111.6 1.9 Leukocyte
IFN 109.4 1.4 IFN.alpha. 16 105.7 1.4 IFN.alpha. 10 96.7 5.5
IFN.alpha. 8 87.5 2.6 IFN.alpha. 5 105.1 3.9 IFN.alpha. 14 100.3
1.4 IFN.alpha. 17 99.8 2.4 IFN.alpha. 7 102.8 3.2 IFN.alpha. 4
100.5 2.5 IFN.alpha. 21 104.4 2.3 IFN-beta 53.0 1.7 IFN-omega 107.1
1.3
Example 6
Inhibition of IFN-Induced IP-10 Secretion by Anti-IFNAR-1
Antibodies
[0376] The addition of IFN alpha 2b to cell culture media has been
shown to induce IP-10 secretion by normal peripheral blood
mononuclear cells (PBMNC). The activity of human anti-IFNAR-1
antibody 3F11 was tested for inhibition of interferon induced
secretion of IP-10 by normal PBMNC cultures by an ELISA binding
assay.
[0377] PBMNC's were incubated in culture medium (RPMI 1640+10%
FBS+1% human serum) with leukocyte IFN, IFN alpha 2b, or IFN
.omega. for 24-48 hours. Supernatants were collected and analyzed
for IP-10/CXCL10 concentration using a quantitative sandwich ELISA
kit (Quantikine.RTM., R&D Systems) at a 1:30 dilution according
to manufacturer recommendations. The results demonstrated that the
human monoclonal antibody 3F11 inhibits leukocyte IFN, recombinant
IFN.alpha. 2b, and recombinant IFN.omega. induced secretion of
IP-10 by normal PBMNC culture. These results are shown in Table
4.
TABLE-US-00004 TABLE 4 Antibody Inhibition of IFN-Induced IP-10
Expression on Normal PBMNC No IFN IFN alpha 2b IFN omega IP-10
Leukocyte IFN IP-10 IP-10 Ab Treatment (pg/ml) IP-10 (pg/ml)
(pg/ml) (pg/ml) No antibody 907 2665 2739 2904 3F11 (5 .mu.g/ml)
387 854 745 674 Control Ig 838 3512 3117 3960 (5 .mu.g/ml) * 100
U/ml of each IFN subtype was added to the cultures
Example 7
Anti-IFNAR-1 Human Monoclonal Antibodies Cross Competition
Assay
[0378] To evaluate whether the human monoclonal antibodies bind to
the same epitope as the mouse 64G12 monoclonal antibody, a
cross-competition ELISA assay was used to determine whether the
antibodies competed for the same binding epitope.
[0379] 96-well plates were coated with soluble CHO-derived human
IFNAR-1 at a concentration of 1 .mu.g/mL in freshly prepared DPBS
at 100 .mu.l/well (Mediatech). Human monoclonal antibodies 3F11,
4G5, 11E2, and 9D4 were added at 20 .mu.g/mL to the wells column 1
and serially diluted at a 1:2 ratio in the wells from column 1 to
column 12, followed by incubation for 45 minutes. Mouse monoclonal
antibody 64G12, at an EC.sub.75 concentration of 0.3 .mu.g/mL, was
added at 50 .mu.L per well and the plates were incubated for 30
minutes. The plates were washed 3 times with Elx 405 auto plate
washer (BIO-TEK Instruments). A peroxidase affinity purified
F(ab')2 goat anti-mouse IgG (Fc.gamma. specific) antibody was
diluted 1:3000 in PBS and added as the detection conjugate (Jackson
ImmunoResearch Laboratories, cat. 115-036-0710), After a one hour
incubation, the plates were washed 3 times with Elx 405 auto plate
washer. An ABTS solution (800 .mu.l ABTS stock, 8 .mu.l 30%
H.sub.2O.sub.2, and 100 mL citrate phosphate buffer) at 27.8 mg/mL
was added to each well and incubated for 20 minutes. The plates
were read at 415 nm using 490 nm as a reference wavelength. The
results are shown in FIG. 11. The results demonstrate that the
human anti-IFNAR-1 monoclonal antibodies, 3F11, 4G5, 11E2, and 9D4
do not compete with 64G12 for binding to IFNAR-1 and thus bind to a
different epitope on IFNAR-1 than 64G12.
Example 8
Antibody Inhibition of SLE Plasma Mediated Dendritic Cell
Development
[0380] SLE plasma induces dendritic cell development from normal
human monocytes. In this example, the purified monoclonal human
anti-IFNAR-1 antibody, 3F11, was tested for inhibition of dendritic
cell development, as assessed by the ability of the antibodies to
inhibit the induction of the cell surface markers CD38, MHC Class I
and CD123 by SLE plasma.
[0381] A 25 ml buffy coat was diluted four fold with PBS. The
sample was separated into 4.times.50ml conical tubes, and 15 ml of
lymphocyte separation medium (ICN Biomedicals) was layered
underneath. Following a 30-minute spin at 500.times.g, the buffy
layer containing the PBMCs was removed and washed with PBS. Cells
were resuspended in culture media at 4.times.10.sup.6 cells/ml.
Monocytes were isolated by incubating PBMC (2.0.times.10.sup.7
cells/5 ml/25 cm.sup.2 flask) for 1.5 hrs at 37.degree. C. in
culture medium and then washing away non-adherent cells twice.
Following the second wash the cells were cultured in media
containing 1% heat inactivated human serum. Twenty five percent SLE
patient plasma plus/minus neutralizing antibodies and isotype
controls (30 ug/ml) were added to the culture flasks; IFN alpha 2b
(100 & 10 iu/ml) plus 25% normal human plasma was used as a
positive control for marker induction. Flasks were incubated at
37.degree. C., 5% CO.sub.2 for three to seven days. Conditioned
medium was harvested from each flask and suspension cells were
recovered by centrifugation at 1000 rpm on a Sorvall RTH-750 rotor.
The pelleted cells were retained on ice and supernate was frozen at
-80.degree. C. for ELISA. Adherent cells were recovered from the
flask with a PBS wash (2 ml), followed by 15 minute incubation in
versene (3 ml), if necessary. The flask was scraped at the end of
the versene incubation and the flask was finally rinsed with PBS
wash (2 ml). Each of the PBS washes and the versene was combined
with the cells recovered from conditioned medium harvest. The
pooled cell suspension was centrifuged at 1000 rpm on a Sorvall
RTH-750 rotor, the resulting pellet was resuspensed to 300 ul in
staining buffer (PBS+0.1M EDTA+2% FBS+1% HS) and dispensed 100
ul/well into a V-bottom 96-well plate. The plate was
pulse-centrifuged at 2800 rpm on a Sorvall RTH-750 rotor and
pelleted cells were resuspended 25 .mu.l/well in flurochrome
labeled antibodies as follows: (1) mouse anti-MHC I-FITC+mouse
anti-CD38-PE, and (2) isotype controls, mouse IgG-FITC+mouse
IgG-PE. The plate was incubated on ice for 45 minutes, protected
from light. The cells were washed three times with the addition of
200 ul staining buffer followed by pulse-celtrifugation and finally
resuspended in 200 .mu.l of 2% paraformaldehyde in PBS. Staining of
dendritic cells was analyzed by flow cytometry with the Becton
Dickinson FACScalibur.TM.. Gates were drawn on the Forward Scatter
vs. Side Scatter graph to remove contaminating cells from the
analysis. The anti-IFNAR-1 human monoclonal antibody 3F11 inhibits
the IFN alpha dependent process of dendritic cell development, as
demonstrated by normalized expression of cell surface markers MHC
Class I, CD38, and CD123 in the presence of 3 F11. The results are
shown below in Table 5, wherein (A) and (B) summarize results for
two representative SLE donor samples.
TABLE-US-00005 TABLE 5 Inhibition of Dendritic Cell Maturation (A)
Donor Plasma #40* (13.3 iU/mL**) Culture Conditions MHC class I
CD123 CD38 MFI MFI MFI 0 IFN/mL 148 14 40 10 IFN/mL 200 19 44 100
IFN/mL 229 26 63 0 206 22 47 3F11 115 13 32 HuIgG1 (isotype 194 22
62 control) (B) Donor Plasma #59* (75.3 iU/mL**) Culture Conditions
MHC class I CD123 CD38 0 IFN/mL 229 11 58 10 IFN/mL 271 12 86 100
IFN/mL 294 13 112 0 202 15 62 3F11 112 8 22 HuIgG1 (isotype 266 14
55 control)
Example 9
Scatchard Binding Analysis of Anti-IFNAR-1 Human Antibodies to
Daudi Cells or Human Peripheral Blood Mononuclear Cells
[0382] Human peripheral blood mononuclear cells were prepared from
fresh blood by standard protocols using Ficol separation. Daudi
cells were obtained from ATCC and grown in RPMI containing 10%
fetal bovine serum (FBS). The cells were washed twice with RPMI
containing 10% FBS at 4 degrees and the cells were adjusted to
4.times.10.sup.7 cells/ml in RPMI media containing 10% fetal bovine
serum (binding buffer). Millipore plates (MAFB NOB) were coated
with 1% nonfat dry milk in water and stored a 4.degree. C.
overnight. The plates were washed with binding buffer and 25 ul of
unlabeled antibody (1000-fold excess) in binding buffer was added
to control wells in a Millipore 96 well glass fiber filter plate
(non-specific binding NSB). Twenty-five microliters of buffer alone
was added to the maximum binding control well (total binding).
Twenty-five microliters of varying concentration of
.sup.125I-anti-IFNAR-1 antibody and 25 ul of Daudi cells or human
peripheral blood mononuclear cells (4.times.10.sup.7 cells/ml) in
binding buffer were added. The plates were incubated for 2 hours at
200 RPM on a shaker at 4.degree. C. At the completion of the
incubation the Millipore plates were washed twice with 0.2 ml of
cold binding buffer. The filters were removed and counted in a
gamma counter. Evaluation of equilibrium binding was performed
using single site binding parameters with the Prism software (San
Diego, Calif.).
[0383] Using the above Scatchard binding assay, the K.sub.D of the
antibody for Daudi cells and for human peripheral blood mononuclear
cells was approximately 0.2 nM and 0.5 nM, respectively.
TABLE-US-00006 SUMMARY OF SEQUENCE LISTING SEQ ID NO: SEQUENCE 1 VH
CDR1 a.a. 3F11 2 VH CDR1 a.a. 4G5 3 VH CDR1 a.a. 11E2 4 VH CDR1
a.a. 9D4 5 VH CDR2 a.a. 3F11 6 VH CDR2 a.a. 4G5 7 VH CDR2 a.a. 11E2
8 VH CDR2 a.a. 9D4 9 VH CDR3 a.a. 3F11 10 VH CDR3 a.a. 4G5 11 VH
CDR3 a.a. 11E2 12 VH CDR3 a.a. 9D4 13 VK CDR1 a.a. 3F11 14 VK CDR1
a.a. 4G5 15 VK CDR1 a.a. 11E2 16 VK CDR1 a.a. 9D4 17 VK CDR2 a.a.
3F11 18 VK CDR2 a.a. 4G5 19 VK CDR2 a.a. 11E2 20 VK CDR2 a.a. 9D4
21 VK CDR3 a.a. 3F11 22 VK CDR3 a.a. 4G5 23 VK CDR3 a.a. 11E2 24 VK
CDR3 a.a. 9D4 25 VH a.a. 3F11 26 VH a.a. 4G5 27 VH a.a. 11E2 28 VH
a.a. 9D4 29 VK a.a. 3F11 30 VK a.a. 4G5 31 VK a.a. 11E2 32 VK a.a.
9D4 33 VH n.t. 3F11 34 VH n.t. 4G5 35 VH n.t. 11E2 36 VH n.t. 9D4
37 VK n.t. 3F11 38 VK n.t. 4G5 39 VK n.t. 11E2 40 VK n.t. 9D4 41 VH
4-34 germline a.a. 42 VH 5-51 germline a.a. 43 VK L18 germline a.a.
44 VK A27 germline a.a.
Sequence CWU 1
1
4415PRTHomo sapiens 1Gly Tyr Phe Trp Ser1 525PRTHomo sapiens 2Asn
Tyr Tyr Trp Ser1 535PRTHomo sapiens 3Asn Tyr Trp Ile Ala1
545PRTHomo sapiens 4Asn Tyr Trp Ile Ala1 5516PRTHomo sapiens 5Glu
Ile Asp His Ser Gly Lys Thr Asn Tyr Asn Pro Ser Leu Lys Ser1 5 10
15616PRTHomo sapiens 6Glu Ile Ile Leu Ser Gly Ser Thr Asn Tyr Asn
Pro Ser Leu Lys Ser1 5 10 15717PRTHomo sapiens 7Ile Ile Tyr Pro Gly
Asp Ser Asp Ile Arg Tyr Ser Pro Ser Phe Gln1 5 10 15Gly817PRTHomo
sapiens 8Ile Ile Tyr Pro Gly Asp Ser Asp Ile Arg Tyr Ser Pro Ser
Phe Gln1 5 10 15Gly910PRTHomo sapiens 9Glu Ser Lys Tyr Tyr Phe Gly
Leu Asp Val1 5 101010PRTHomo sapiens 10Glu Ser Lys Trp Gly Tyr Tyr
Phe Asp Ser1 5 10118PRTHomo sapiens 11His Asp Ile Glu Gly Phe Asp
Tyr1 5128PRTHomo sapiens 12His Asp Ile Glu Gly Phe Asp Tyr1
51311PRTHomo sapiens 13Arg Ala Ser Gln Gly Ile Tyr Ser Val Leu Ala1
5 101411PRTHomo sapiens 14Arg Ala Thr Gln Asp Ile Ser Ile Ala Leu
Val1 5 101512PRTHomo sapiens 15Arg Ala Ser Gln Ser Val Ser Ser Ser
Phe Phe Ala1 5 101612PRTHomo sapiens 16Arg Ala Ser Gln Ser Val Ser
Ser Ser Phe Phe Ala1 5 10177PRTHomo sapiens 17Asp Ala Ser Arg Leu
Glu Ser1 5187PRTHomo sapiens 18Asp Ala Ser Gly Leu Gly Ser1
5197PRTHomo sapiens 19Gly Ala Ser Ser Arg Ala Thr1 5207PRTHomo
sapiens 20Gly Ala Ser Ser Arg Ala Thr1 5218PRTHomo sapiens 21Gln
Gln Phe Asn Ser Tyr Ile Thr1 5229PRTHomo sapiens 22Gln Gln Phe Asn
Ser Tyr Pro Tyr Thr1 5239PRTHomo sapiens 23Gln Gln Tyr Asp Ser Ser
Ala Ile Thr1 5249PRTHomo sapiens 24Gln Gln Tyr Asp Ser Ser Ala Ile
Thr1 525118PRTHomo sapiens 25Gln Val Gln Leu Gln Gln Trp Gly Ala
Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val
Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30Phe Trp Ser Trp Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Asp His Ser
Gly Lys Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile
Ser Val Asp Thr Ser Lys Asn Gln Val Ser Leu65 70 75 80Lys Leu Ser
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Glu
Ser Lys Tyr Tyr Phe Gly Leu Asp Val Trp Gly Gln Gly Thr 100 105
110Thr Val Thr Val Thr Ser 11526118PRTHomo sapiens 26Gln Val Gln
Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu
Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asn Tyr 20 25 30Tyr
Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45Gly Glu Ile Ile Leu Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys
50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser
Leu65 70 75 80Asn Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95Arg Glu Ser Lys Trp Gly Tyr Tyr Phe Asp Ser Trp
Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser 11527117PRTHomo
sapiens 27Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ile Phe
Thr Asn Tyr 20 25 30Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly
Leu Glu Ser Met 35 40 45Gly Ile Ile Tyr Pro Gly Asp Ser Asp Ile Arg
Tyr Ser Pro Ser Phe 50 55 60Gln Gly Gln Val Thr Ile Ser Ala Asp Lys
Ser Ile Thr Thr Ala Tyr65 70 75 80Leu Gln Trp Ser Ser Leu Lys Ala
Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg His Asp Ile Glu Gly
Phe Asp Tyr Trp Gly Arg Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11528117PRTHomo sapiens 28Glu Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser
Gly Tyr Ile Phe Thr Asn Tyr 20 25 30Trp Ile Ala Trp Val Arg Gln Met
Pro Gly Lys Gly Leu Glu Ser Met 35 40 45Gly Ile Ile Tyr Pro Gly Asp
Ser Asp Ile Arg Tyr Ser Pro Ser Phe 50 55 60Gln Gly Gln Val Thr Ile
Ser Ala Asp Lys Ser Ile Thr Thr Ala Tyr65 70 75 80Leu Gln Trp Ser
Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg His
Asp Ile Glu Gly Phe Asp Tyr Trp Gly Arg Gly Thr Leu 100 105 110Val
Thr Val Ser Ser 11529106PRTHomo sapiens 29Ala Ile Gln Leu Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Gly Ile Tyr Ser Val 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Thr Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala
Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Ile Thr
85 90 95Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 10530107PRTHomo
sapiens 30Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Thr Gln Asp Ile
Ser Ile Ala 20 25 30Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Glu Leu Leu Ile 35 40 45Tyr Asp Ala Ser Gly Leu Gly Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Phe Asn Ser Tyr Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys 100 10531108PRTHomo sapiens 31Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30Phe Phe Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr
Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Leu Ser 50 55 60Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asp Ser Ser Ala
85 90 95Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100
10532108PRTHomo sapiens 32Glu Ile Val Leu Thr Gln Ser Pro Gly Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser Ser Ser 20 25 30Phe Phe Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg
Ala Thr Gly Ile Pro Asp Arg Leu Ser 50 55 60Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Thr Arg Leu Glu65 70 75 80Pro Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Tyr Asp Ser Ser Ala 85 90 95Ile Thr Phe
Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 10533354DNAHomo sapiens
33caggtgcagc tacagcagtg gggcgcagga ctgttgaagc cttctgagac cctgtccctc
60acctgcgctg tctatggtgg gtccttcagt ggttatttct ggagctggat ccgccagccc
120ccagggaagg ggctggagtg gattggggaa atcgatcaca gtggaaagac
caactacaat 180ccgtccctca agagtcgagt taccatatca gtagacacgt
ccaagaacca ggtctccctg 240aagctgagct ctgtgaccgc cgcggacacg
gctgtgtatt actgtgcgag agaaagcaag 300tactacttcg gtttggacgt
ctggggccaa gggaccacgg tcaccgtcac ctca 35434354DNAHomo sapiens
34caggtgcagc tacagcagtg gggcgcagga ctgttgaagc cttcggagac cctgtccctc
60acctgcgctg tctatggtgg gtccttcagt aattactact ggagctggat ccgccagccc
120ccagggaagg ggctggagtg gattggggaa atcattctta gtggaagcac
caactacaac 180ccgtccctca agagtcgagt caccatatca gtagacacgt
ccaagaacca gttctccctg 240aacctgacct ctgtgaccgc cgcggacacg
gctgtgtatt actgtgcgag agagtctaaa 300tggggttact actttgactc
ctggggccag ggaaccctgg tcaccgtctc ctca 35435351DNAHomo sapiens
35gaggtgcagc tggtgcagtc tggagcagag gtgaaaaagc ccggggagtc tctgaagatc
60tcctgtaagg gttctggata catctttacc aattactgga tcgcctgggt gcgccagatg
120cccggtaaag gcctggagtc gatggggatc atctatcctg gtgactctga
tatcagatac 180agcccgtcct tccaaggcca ggtcaccatc tcagccgaca
agtccatcac caccgcctac 240ctgcagtgga gcagtctgaa ggcctcagac
accgccatgt attactgtgc gagacatgac 300atagaggggt ttgactactg
gggccgggga accctggtca ccgtctcctc a 35136318DNAHomo sapiens
36gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc
60atcacttgcc gggcaagtca gggcatttac agtgttttag cctggtatca gcagaaacca
120gggaaaactc ctaagctcct gatctatgat gcctcccgtt tggaaagtgg
ggtcccatca 180aggttcagcg gcagtggatc tgggacagat ttcactctca
ccatcagcag cctgcagcct 240gaagattttg caacttatta ctgtcaacag
tttaatagtt acatcacctt cggccaaggg 300acacgactgg agattaaa
31837351DNAHomo sapiens 37gaggtgcagc tggtgcagtc tggagcagag
gtgaaaaagc ccggggagtc tctgaagatc 60tcctgtaagg gttctggata catctttacc
aactactgga tcgcctgggt gcgccagatg 120cccggtaaag gcctggagtc
gatggggatc atctatcctg gtgactctga tatcagatac 180agcccgtcct
tccaaggcca ggtcaccatc tcagccgaca agtccatcac caccgcctac
240ctgcagtgga gcagtctgaa ggcctcagac accgccatgt attactgtgc
gagacatgac 300atagaggggt ttgactactg gggccgggga accctggtca
ccgtctcctc a 35138320DNAHomo sapiens 38gccatccagt tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaactca
ggacattagc attgctttag tctggtatca gcagaaacca 120gggaaagctc
ctgagctcct gatctatgat gcctccggtt tgggaagtgg ggtcccatca
180aggttcagcg gcagtggatc tggcacagat ttcactctca ccatcagcag
cctgcagcct 240gaagattttg caacttatta ctgtcaacag ttaatagtta
cccgtacact tttggccagg 300ggaccaagct ggagatcaaa 32039324DNAHomo
sapiens 39gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga
aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcagcttct tcgcctggta
ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca
gcagggccac tggcatccca 180gacaggttaa gtggcagtgg gtctgggaca
gacttcactc tcaccatcac cagactggag 240cctgaagatt ttgcagtgta
ttactgtcag cagtatgata gctcagcgat caccttcggc 300caagggacac
gactggagat taaa 32440324DNAHomo sapiens 40gaaattgtgt tgacgcagtc
tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca
gagtgttagc agcagcttct tcgcctggta ccagcagaaa 120cctggccagg
ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca
180gacaggttaa gtggcagtgg gtctgggaca gacttcactc tcaccatcac
cagactggag 240cctgaagatt ttgcagtgta ttactgtcag cagtatgata
gctcagcgat caccttcggc 300caagggacac gactggagat taaa 3244197PRTHomo
sapiens 41Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro
Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe
Ser Gly Tyr 20 25 30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly
Leu Glu Trp Ile 35 40 45Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr
Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser
Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg4298PRTHomo sapiens
42Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1
5 10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser
Tyr 20 25 30Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu
Trp Met 35 40 45Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser
Pro Ser Phe 50 55 60Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile
Ser Thr Ala Tyr65 70 75 80Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp
Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg4395PRTHomo sapiens 43Ala
Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala
20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe
Asn Ser Tyr Pro 85 90 954495PRTHomo sapiens 44Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Gly
Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90
95
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References